Methods, kits and compositions pertaining to combination oligomers and libraries for their preparation

ABSTRACT

This invention pertains to the field of combination oligomers, including the block synthesis of combination oligomers in the absence of a template, as well as related methods, kits, libraries and other compositions.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.10/096,125, filed Mar. 9, 2002 incorporated herein by reference, whichapplication claims the benefit of U.S. Provisional Patent ApplicationSer. No. 60/274,547 filed on Mar. 9, 2001, incorporated herein byreference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention pertains to the field of combination oligomers, includingthe block synthesis of combination oligomers in the absence of atemplate, as well as methods, kits, libraries and other compositions.

2. Introduction

Nucleic acid hybridization is a fundamental process in molecularbiology. Probe-based assays are useful in the detection, quantitationand/or analysis of nucleic acids. Nucleic acid probes have long beenused to analyze samples for the presence of nucleic acid from bacteria,fungi, virus or other organisms and are also useful in examininggenetically-based disease states or clinical conditions of interest.Nonetheless, nucleic acid probe-based assays have been slow to achievecommercial success. This lack of commercial success is, at leastpartially, the result of difficulties associated with specificity,sensitivity and/or reliability.

Nucleic acid amplification assays comprise an important class ofspecific target sequence detection methods in modern biology, withdiverse applications in diagnosis of inherited disease, humanidentification, identification of microorganisms, paternity testing,virology, and DNA sequencing. The polymerase chain reaction (PCR)amplification method allows for the production and detection of targetnucleic acid sequences with great sensitivity and specificity. PCRmethods are integral to cloning, analysis of genetic expression, DNAsequencing, genetic mapping, drug discovery, and the like (Gilliland,Proc. Natl. Acad. Sci., 87: 2725-2729 (1990); Bevan, PCR Methods andApplications 1: 222-228 (1992); Green, PCR Methods and Applications, 1:77-90 (1991); McPherson, M. J., Quirke, P., and Taylor, G. R. in PCR 2:A Practical Approach Oxford University Press, Oxford (1995)). Methodsfor detecting a PCR product (amplicon) using an oligonucleotide probecapable of hybridizing with the target sequence or amplicon aredescribed in Mullis, U.S. Pat. Nos. 4,683,195 and 4,683,202; EP No.237,362.

Despite its name, Peptide Nucleic Acid (PNA) is neither a peptide, anucleic acid nor is it an acid. Peptide Nucleic Acid (PNA) is anon-naturally occurring polyamide (pseudopeptide) that can hybridize tonucleic acid (DNA and RNA) with sequence specificity (See: U.S. Pat. No.5,539,082 and Egholm et al., Nature 365: 566-568 (1993)). PNA has beencharacterized in the scientific literature as a nucleic acid mimic,rather than a nucleic acid analog, since its structure is completelysynthetic and not derived from nucleic acid (See: Nielsen, P. E., Acc.Chem. Res. 32: 624-630 (1999)).

Being a non-naturally occurring molecule, unmodified PNA is not known tobe a substrate for the enzymes that are known to degrade peptides ornucleic acids. Therefore, PNA should be stable in biological samples, aswell as have a long shelf-life. Unlike nucleic acid hybridization, whichis very dependent on ionic strength, the hybridization of a PNA with anucleic acid is fairly independent of ionic strength and is favored atlow ionic strength, conditions that strongly disfavor the hybridizationof nucleic acid to nucleic acid (Egholm et al., Nature, at p. 567).Because of their unique properties, it is clear that PNA is not theequivalent of a nucleic acid in either structure or function.

In addition to the limitations of selectivity/discrimination, there is aneed to be able to rapidly and efficiently prepare numerous oligomersthat can be used as probes or primers in an assay at a defined scalethat is small enough to be cost effective. This need has arisen becausegenome sequencing has provided massive amounts of raw sequence data thatcan be mined for useful information. Because the volume of informationis so massive, screening results that are generated from the miningoperations typically involve high throughput analysis that requires tensor even hundreds of thousands of probes and primers. However,commercially available instruments that build nucleic acid and peptidenucleic acids based upon stepwise monomer assembly (de novo synthesis)require hours to produce single probes in a scale that is costprohibitive for the manufacture of thousands or tens of thousands ofprobes or primers. Moreover, because the probes are synthesized de novo,it is difficult to expedite the delivery of the tens to hundreds ofthousands of probes or primers within a short period, for example sixmonths or less, without a massive investment in capital equipment.Therefore, it would be advantageous to be able to have a method for therapid (days or weeks), efficient and cost effective production of tensor even hundreds of thousands of oligomers of desired nucleobasesequence that could be used as probes or primers for high throughputapplications such as for expression analysis or the mining of genomes.

SUMMARY OF THE INVENTION

This invention pertains to the field of combination oligomers, includingthe block synthesis of combination oligomers in the absence of atemplate, as well as related methods, kits, libraries and othercompositions. As used herein a combination oligomer is a compositioncomprising two or more oligomer blocks independently selected frompeptide nucleic acid, PNA chimera and PNA combination oligomer, whetheror not labeled, wherein said oligomer blocks are linked by a linker.This linker can optionally be a cleavage site for an enzyme. Theaforementioned composition is referred to herein as a combinationoligomer, without regard to its method of production, because itshybridization properties result from the combined properties of the twocomponent oligomer blocks as well as the nature of the linker and theopportunity for interaction between the blocks when the combinationoligomer hybridizes to a target sequence.

In one embodiment, this invention pertains to novel combinationoligomers. The novel combination oligomers of this invention arereferred to herein as self-indicating combination oligomers andsubstrate combination oligomers; each of which is described in moredetail below (See also: Examples 4 & 5, below). It is to be understoodthat combination oligomers can be used as probes or primers in numerousapplications. The requirement for probes is merely that they hybridizeto a target sequence with sequence specificity. Thus, when used as aprobe, there are no additional limitations on specific features of thecombination oligomer. However, when used as a primer, it is arequirement that the combination oligomer contain moieties suitable forthe recognition and operation of an enzyme since polymerase enzymes arenot known to operate on unmodified PNA oligomers (See: Lutz et al., J.Am. Chem. Soc. 119: 3177-3178 (1997)).

In another embodiment, this invention pertains to a compositioncomprising a polynucleobase strand and a combination oligomer sequencespecifically hybridized to a target sequence of contiguous nucleobaseswithin the polynucleobase strand to thereby form a double strandedtarget sequence/combination oligomer complex (See FIG. 1; Also note thatthis configuration is sometimes referred to herein as being hybridizedjuxtaposed to the target sequence such that there is no gap or gapbase). The combination oligomer comprises a first and a second oligomerblock that are each independently a peptide nucleic acid, PNA chimera orPNA combination oligomer. The first and second oligomer blocks arecovalently linked by a linker of at least three atoms in length.

In yet another embodiment, this invention pertains to a method fordetermining a target sequence of contiguous nucleobases. The methodcomprises contacting the target sequence with a combination oligomer,under suitable hybridization conditions, wherein the combinationoligomer comprises a first oligomer block and a second oligomer blockthat are each independently a peptide nucleic acid, PNA chimera or PNAcombination oligomer. The first and second oligomer blocks are linkedcovalently to each other by a linker that is at least three atoms inlength. Moreover, the first and second oligomer blocks can sequencespecifically hybridize to the target sequence of contiguous nucleobasesto thereby form a double stranded target sequence/combination oligomercomplex. Complex formation is determined to thereby determine the targetsequence since the complex does not form in the absence of the targetsequence. Determination of the complex includes, but is not limited to,determining the presence, absence, quantity (amount) or position of thecomplex to thereby determine the presence, absence, quantity (amount),position or identity of the target sequence (See for example: Examples3-5).

In still another embodiment, this invention pertains to a method fordetermining the zygosity of a nucleic acid for a single nucleotidepolymorphism (SNP). The method comprises contacting a nucleic acidsample with at least two independently detectable combination oligomers.Each independently detectable combination oligomer comprises a firstoligomer block and a second oligomer block that are each independently apeptide nucleic acid, PNA chimera or PNA combination oligomer and theoligomer blocks are linked covalently to each other by a linker that isat least three atoms in length. The first and second oligomer blockstaken together encode a probing nucleobase sequence that is designed tosequence specifically hybridize to a target sequence of contiguousnucleobases in a polynucleobase strand of the nucleic acid sample, ifpresent, to thereby form a double stranded target sequence/independentlydetectable combination oligomer complex. The probing nucleobase sequencein each independently detectable combination oligomer differs from theother by at least one nucleobase (the SNP nucleobase). However, theprobing nucleobase sequence may differ by more than one nucleobase,depending on probe design, provided however that they differ by a singlenucleobase at the SNP to be determined (See for example: Example 5,Table 9 and particularly the sets of PNA combination oligomer probes forSNPs 6876 and 6879).

According to the method, the nucleic acid sample and combinationoligomers are contacted with one or more reagents suitable forperforming a nucleic amplification reaction that amplifies the nucleicacid present in the sample and nucleic acid amplification is performedin the presence of the nucleic acid, the combination oligomers and thereagents. Complex formation for each independently detectablecombination oligomer/target sequence complex is determined to therebydetermine whether the nucleic acid is heterozygous or homozygous for aparticular SNP. Complex determination can be correlated with thezygosity state of a particular SNP, since the complexes will not form inthe absence of the respective target sequence of contiguous nucleobasesfor each particular combination oligomer. Moreover, the twoindependently detectable combination oligomers provide all of theinformation needed to determine the three possible genotype statesdepending on which complexes do and do not form.

In one embodiment, the independently detectable combination oligomersare independently detectable, self-indicating combination oligomers.According to the method a determination, under suitable hybridizationconditions, is made of any change in detectable signal arising from atleast one of the labels of each of the independently detectable energytransfer sets as a measure of whether or not each of the combinationoligomers is hybridized to their respective target sequence ofcontiguous nucleobases. Such determination can be performed eitherduring the process of the nucleic acid amplification (e.g. in real-time)or after the nucleic acid amplification reaction is completed (e.g. atthe end-point). According to the method, the result of the change insignal for at least one label of each energy transfer set of eachcombination oligomer is correlated with a determination of the formationof each of the two possible target sequence/independently detectableself-indicating combination oligomer complexes. Based upon this data,one of the three possible states of zygosity of the sample for aparticular SNP can be determined (See for example: Example 5).

In another embodiment, this invention is directed to a method forforming a combination oligomer from oligomer blocks. The methodcomprises reacting a first oligomer block, a second oligomer block, andoptionally a condensation reagent or reagents under condensationconditions to thereby form a combination oligomer having a linker of atleast three atoms in length that covalently links the first oligomerblock to the second oligomer block. According to the method, the firstand second oligomer blocks are each independently a peptide nucleicacid, PNA chimera or PNA combination oligomer. Neither of the first orsecond oligomer blocks is support bound and the combination oligomerforms in the absence of a template. The ligation/condensation reactioncan be performed in aqueous solution. The nucleobases need not beprotected during the condensation/ligation reaction.

In yet another embodiment, this invention pertains to another method forforming combination oligomers from oligomer blocks. The method comprisesreacting a first oligomer block, a second oligomer block, and optionallya condensation reagent or reagents under condensation conditions tothereby form a combination oligomer having a linker of at least threeatoms in length that covalently links the first oligomer block to thesecond oligomer block. The first and second oligomer blocks are eachindependently a peptide nucleic acid, PNA chimera or PNA combinationoligomer. The nucleobases of the oligomer blocks do not compriseprotecting groups and the combination oligomer forms in the absence of atemplate. The ligation/condensation can be performed in aqueoussolution.

Regardless of the method of forming a combination oligomer as describedabove, in another embodiment, the product of the condensation/ligationreaction can optionally be further lengthened/elongated. Hence, thecombination oligomer, as formed, can be used as an oligomer block suchthat repeating the method produces a further lengthened/elongatedoligomer. According to the method, the combination oligomer, aspreviously formed, can optionally be deprotected, as may be required, tofacilitate the next condensation/ligation step. The combinationoligomer, as previously formed and optionally deprotected, is reactedwith a third oligomer block and optionally a condensation reagent orreagents under condensation conditions. This forms the elongatedcombination oligomer having a covalent linkage of at least three atomsin length that covalently links the third oligomer block to thecombination oligomer wherein, the elongated combination oligomer formsin the absence of a template. In accordance with this method, thisprocess can be optionally repeated until the combination oligomer is ofthe desired length. Such a process of continued elongation can, forexample, be useful for the preparation of arrays since longer oligomersare often used for this application.

In certain other embodiments of this invention, a combination oligomeris formed that possesses a cleavage site for an enzyme wherein thecleavage site is protected from cleavage upon the binding of thecombination oligomer to a binding pair. Hence, this invention alsopertains to a method for determining whether or not a combinationoligomer binds to a possible binding partner (e.g. an aptmer or targetsequence). The method comprises contacting the combination oligomer andthe possible binding partner under suitable binding conditions tothereby possibly form a combination oligomer/binding partner complex.According to the method the combination oligomer is a polymer comprisinga segment of the formula: A-W-C, wherein A and C are oligomer blocksthat are optionally linked to other moieties and that are eachindependently a peptide nucleic acid, PNA chimera or PNA combinationoligomer. The group W is a linker of at least three atoms in length thatcovalently links oligomer block A to oligomer block C and that is acleavage site for an enzyme.

According to the method the binding partner and the combination oligomerare treated with an enzyme suitable for cleaving the cleavage site undersuitable enzyme cleaving conditions. Then a determination is made ofwhether or not the combination oligomer has been cleaved by the activityof the enzyme to thereby determine whether or not the combinationoligomer/binding partner complex formed.

Where the binding partner is a target sequence, and the combinationoligomer is bound to the target sequence, it is protected from theactivity of the enzyme. Accordingly, if the assay determines that thecombination oligomer is not substantially degraded, it must have boundto the target sequence (See: Example 4). Conversely, where thecombination oligomer was not protected from degradation, it can beconcluded that the target sequence was not present. It is also to beunderstood that since such an assay relies upon an enzymatic event,quantitation of the target sequence can be determined by determiningenzyme activity.

In still another embodiment, this invention pertains to kits. In oneembodiment said kit comprises two or more independently detectablecombination oligomers wherein each of said independently detectablecombination oligomers comprises a first oligomer block and a secondoligomer block that are each independently a peptide nucleic acid, a PNAchimera or PNA combination oligomer. The oligomer blocks are linkedcovalently to each other by a linker that is at least three atoms inlength. In each independently detectable combination oligomer, the firstand second oligomer blocks taken together encode a probing nucleobasesequence that is designed to sequence specifically hybridize to a targetsequence of contiguous nucleobases that is suitable for the formation ofa double stranded target sequence/combination oligomer complex. Theprobing nucleobase sequence in each independently detectable combinationoligomer differs from the probing nucleobase sequences of the otherindependently detectable combination oligomer(s) by at least onenucleobase. Each independently detectable combination oligomer containsat least one independently detectable label. The kit optionallycomprises; (i) one or more oligonucleotides; (ii) one or more buffers;(iii) one or more nucleotide triphosphates; (iv) a nucleic acidamplification master mix; or (v) one or more polymerase enzymes.

In yet another embodiment, this invention pertains to a set of two ormore independently detectable combination oligomers. The combinationoligomers of the set each comprise a first oligomer block and a secondoligomer block that are each independently a peptide nucleic acid, PNAchimera or PNA combination oligomer. The oligomer blocks are linkedcovalently to each other by a linker that is at least three atoms inlength. In each independently detectable combination oligomer, the firstand second oligomer blocks taken together encode a probing nucleobasesequence that is designed to sequence specifically hybridize to a targetsequence of contiguous nucleobases to thereby form a double strandedtarget sequence/combination oligomer complex. The probing nucleobasesequence in each independently detectable combination oligomer differsfrom the probing nucleobase sequences of the other independentlydetectable combination oligomer(s) of the set by at least onenucleobase. Each independently detectable combination oligomer containsat least one independently detectable label.

This invention is also directed to a method for forming a terminaloligomer block and a condensing oligomer block from a bifunctionalsingle set library. The method comprises providing a bifunctional singleset library of at least two oligomer blocks. One oligomer block of thebifunctional single set library is treated to thereby remove one or moreof the protecting groups to thereby produce a terminal oligomer. Oneoligomer block of the bifunctional single set library is also treated toremove one or more different protecting groups, as compared with thosethat produce the terminal oligomer block, to thereby produce acondensing oligomer block.

Thus, this invention is also directed to a compound library comprising abifunctional single set of oligomer blocks. The oligomer blocks of theset can be used to produce both terminal oligomer blocks andcondensation oligomer blocks by the removal of certain protectinggroups. The oligomer blocks of the bifunctional set are peptide nucleicacid oligomer, PNA chimera or PNA combination oligomer. The oligomerblocks of the bifunctional set are selected to comprise functionalmoieties that form a linker of at least three atoms in length when aterminal oligomer block is condensed with a condensation oligomer block.Furthermore, the oligomer blocks are not support bound and do notcomprise nucleobase protecting groups.

In still another embodiment, this invention pertains to another compoundlibrary. According the invention, this compound library comprises atleast one set of terminal oligomer blocks and at least one set ofcondensing oligomer blocks wherein each set of blocks comprises two ormore different oligomers and said oligomer blocks are selected from thegroup consisting of: peptide nucleic acid oligomer, PNA chimera and PNAcombination oligomer. The oligomer blocks are selected to comprisefunctional moieties that form a linker of at least three atoms in lengthwhen a terminal oligomer block is condensed with a condensation oligomerblock. Additionally, the oligomer blocks are not support bound and theoligomer blocks do not comprise nucleobase-protecting groups. It is tobe understood that a compound library of this invention is not to belimited to one or two sets of block oligomers. By way of a non-limitingexample, the library may comprise three or more sets of oligomer blocks.

Accordingly, in still another embodiment, this invention pertains toanother compound library. The compound library comprises at least oneset of terminal oligomer blocks and at least two sets of condensingoligomer blocks. According to the invention, each set of oligomer blockscomprises two or more different oligomers and the oligomer blocks ofeach set are independently either peptide nucleic acid oligomer, PNAchimera or PNA combination oligomer. The oligomer blocks are selected tocomprise functional moieties that form a linker of at least three atomsin length that covalently links the oligomer blocks when a terminaloligomer block is condensed with a condensation oligomer block.Additionally, the oligomer blocks are not support bound and the oligomerblocks do not comprise nucleobase-protecting groups. Furthermore, all ofthe oligomer blocks of a set of condensing oligomer blocks contain thesame independently detectable reporter moiety and all of the oligomerblocks of the at least one set of terminal oligomer blocks comprise thesame quencher moiety.

In still another embodiment, this invention pertains to novelcombination oligomers such as a self-indicating combination oligomer.Accordingly, this invention also pertains to a composition of covalentlylinked oligomer blocks comprising a segment of the formula: A-B-C.According to the invention, oligomer blocks A and C are eachindependently a peptide nucleic acid, PNA chimera or PNA combinationoligomer and are optionally linked to other moieties of the segment. Thelinker B is at least three atoms in length and covalently links oligomerblock A to oligomer block C. Moreover, oligomer blocks A and C takentogether encode a probing nucleobase sequence that is designed tosequence specifically hybridize to a target sequence of contiguousnucleobases to thereby form a double stranded targetsequence/combination oligomer complex.

The self-indicating combination oligomer further comprises an energytransfer set of labels such that at least one acceptor moiety of theenergy transfer set is linked to one of the linked oligomer blocks ofthe composition whilst at least one donor moiety of the energy transferset is linked to another of the linked oligomer blocks of thecomposition wherein labels of the set are linked to the combinationoligomer at positions that facilitate a change in detectable signal ofat least one label when the combination oligomer is sequencespecifically hybridized to a target sequence as compared to when thecombination oligomer is in a non-hybridized state.

In yet another embodiment, this invention pertains to an array of atleast two combination oligomers wherein at least one of the combinationoligomers comprises a segment having the formula: A-B-C. According tothe invention, oligomer blocks A and C are each independently a peptidenucleic acid, PNA chimera or PNA combination oligomer and are optionallylinked to other moieties. The linker B is at least three atoms in lengthand covalently links oligomer block A to oligomer block C. Oligomerblocks A and C taken together encode a probing nucleobase sequence thatis designed to sequence specifically hybridize to a target sequence ofcontiguous nucleobases to thereby form a double stranded targetsequence/combination oligomer complex.

In yet another embodiment, this invention pertains to methods forforming an array of combination oligomers. In one embodiment, the methodcomprises reacting, at a site on a solid carrier, a first oligomerblock, a second oligomer block, and optionally a condensation reagent orreagents under condensation conditions to thereby form a combinationoligomer having a linker of at least three atoms in length thatcovalently links the first oligomer block to the second oligomer block.According to the invention, one of said two oligomer blocks is supportbound. Further, the first and second oligomer blocks are eachindependently a peptide nucleic acid oligomer, PNA chimera or PNAcombination oligomer. Additionally, one or both oligomer blocks do notcomprise nucleobase protecting groups and the combination oligomer formsin the absence of a template. The method further comprises repeating themethod with one or more different oligomer blocks at one or moredifferent sites until the desired array of combination oligomers isconstructed.

In still another embodiment, this invention pertains to another methodfor forming an array of combination oligomers. The method comprisesreacting, at a site on a solid carrier, a functional group of acombination oligomer having a linker of at least three atoms in lengththat covalently links the first oligomer block to the second oligomerblock with a surface functional group to thereby covalently attach thecombination oligomer to the surface. According to the method, the firstand second oligomer blocks of the combination oligomer are eachindependently a peptide nucleic acid oligomer, PNA chimera or PNAcombination oligomer. Moreover, one or both oligomer blocks do notcomprise nucleobase-protecting groups. The method further comprisesrepeating the method for attachment of the combination oligomer with oneor more different combination oligomers at one or more different sitesuntil the desired array of combination oligomers is constructed.

BRIEF DESCRIPTION OF THE DRAWINGS

The skilled artisan will understand that the drawings, described below,are for illustration purposes only. The drawings are not intended tolimit the scope of the present teachings in any way.

FIG. 1 is an illustration of hybrids formed from a combination oligomerand a target sequence, as described in Example 1, that either do or donot comprise a “gap” or “base gap” (that is to say that the oligomerblocks of the combination oligomer do or do not hybridize to a targetsequence of contiguous nucleobases).

FIG. 2 is a color reproduction of digital images taken with a CCD cameraequipped microscope for PNA-FISH analysis of Salmonella bacteria, asdescribed in Example 3.

FIG. 3 is a graphical representation of data associated with the enzymecleavage assay of Example 4.

FIG. 4 is a bar graph of data obtained for SNP 6784 and Coriell Pedigree1333.

FIG. 5 is an allele distribution plot for data obtained for SNP 6784 andCoriell Pedigree 1333.

FIG. 6 is a bar graph of data obtained for SNP 6802 and Coriell Pedigree1333.

FIG. 7 is an allele distribution plot for data obtained for SNP 6802 andCoriell Pedigree 1333.

FIG. 8 is a bar graph of data obtained for SNP 6806 and Coriell Pedigree1333.

FIG. 9 is an allele distribution plot for data obtained for SNP 6806 andCoriell Pedigree 1333.

FIG. 10 is a bar graph of data obtained for SNP 6834 and CoriellPedigree 1331.

FIG. 11 is an allele distribution plot for data obtained for SNP 6834and Coriell Pedigree 1331.

FIG. 12 is a bar graph of data obtained for SNP 6837 and CoriellPedigree 1341.

FIG. 13 is an allele distribution plot for data obtained for SNP 6837and Coriell Pedigree 1341.

FIG. 14 is a bar graph of data obtained for SNP 6848 and CoriellPedigree 1331.

FIG. 15 is an allele distribution plot for data obtained for SNP 6848and Coriell Pedigree 1331.

FIG. 16 is a bar graph of data obtained for SNP 6876 and CoriellPedigree 1333.

FIG. 17 is an allele distribution plot for data obtained for SNP 6876and Coriell Pedigree 1333.

FIG. 18 is a bar graph of data obtained for SNP 6876 and CoriellPedigree 1341.

FIG. 19 is an allele distribution plot for data obtained for SNP 6876and Coriell Pedigree 1341.

FIG. 20 is a bar graph of data obtained for SNP 6879 and CoriellPedigree 1331.

FIG. 21 is an allele distribution plot for data obtained for SNP 6879and Coriell Pedigree 1331.

FIG. 22 is a bar graph of data obtained for SNP 6885 and CoriellPedigree 1333.

FIG. 23 is an allele distribution plot for data obtained for SNP 6885and Coriell Pedigree 1333.

FIG. 24 is a bar graph of data obtained for SNP 6885 and CoriellPedigree 1341.

FIG. 25 is an allele distribution plot for data obtained for SNP 6885and Coriell Pedigree 1341.

FIG. 26 is a bar graph of data obtained for SNP 6885 and CoriellPedigree 1341 using two ligated combination oligomers.

FIG. 27 is an allele distribution plot for data obtained for SNP 6885and Coriell Pedigree 1341 using two ligated combination oligomers.

FIGS. 28 a and 28 b are illustrations of possible ligation reactionswherein one oligomer block comprises an amino group that couples to thecarboxylic acid group of a second oligomer block.

FIGS. 29 a, 29 b and 29 c are illustrations of possible ligationreactions involving borohydride reduction.

FIGS. 30 a and 30 b are illustrations of additional possible ligationreactions involving borohydride reduction.

FIGS. 31 a, 31 b and 31 c are illustrations of possible ligationreactions involving thiol reactive groups.

FIG. 32 contains illustrations non-naturally occurring nucleobases thatcan be used in the oligomer blocks or combination oligomers of thisinvention to produce non-standard base pairing motifs.

FIG. 33 contains an illustration of both Dye 1 and Dye 2.

DETAILED DESCRIPTION OF THE INVENTION

1. Definitions:

For the purposes of interpreting this specification the followingdefinitions shall apply and whenever appropriate, terms used in thesingular shall also include the plural and vice versa.

-   a. As used herein, “nucleobase” means those naturally occurring and    those non-naturally occurring heterocyclic moieties commonly known    to those who utilize nucleic acid technology or utilize peptide    nucleic acid technology to thereby generate polymers that can    sequence specifically bind to nucleic acids. Non-limiting examples    of suitable nucleobases include: adenine, cytosine, guanine,    thymine, uracil, 5-propynyl-uracil, 2-thio-5-propynyl-uracil,    5-methylcytosine, pseudoisocytosine, 2-thiouracil and 2-thiothymine,    2-aminopurine, N9-(2-amino-6-chloropurine), N9-(2,6-diaminopurine),    hypoxanthine, N9-(7-deaza-guanine), N9-(7-deaza-8-aza-guanine) and    N8-(7-deaza-8-aza-adenine). Other non-limiting examples of suitable    nucleobase include those nucleobases illustrated in FIGS. 2(A) and    2(B) of Buchardt et al. (WO92/20702 or WO92/20703).-   b. As used herein, “nucleobase sequence” means any segment, or    aggregate of two or more segments (e.g. the aggregate nucleobase    sequence of two or more oligomer blocks), of a polymer that    comprises nucleobase-containing subunits. Non-limiting examples of    suitable polymers or polymers segments include oligodeoxynucleotides    (e.g. DNA), oligoribonucleotides (e.g. RNA), peptide nucleic acids    (PNA), PNA chimeras, PNA combination oligomers, nucleic acid analogs    and/or nucleic acid mimics.-   c. As used herein, “target sequence” is a nucleobase sequence of a    polynucleobase strand sought to be determined. It is to be    understood that the nature of the target sequence is not a    limitation of this invention. The polynucleobase strand comprising    the target sequence may be provided from any source. For example,    the target sequence may exist as part of a nucleic acid (e.g. DNA or    RNA), PNA, nucleic acid analog or other nucleic acid mimic. The    sample containing the target sequence may be provided from nature or    it may be synthesized or supplied from a manufacturing process. When    the target sequence is a subsequence of a nucleic acid, said nucleic    acid can be obtained from any source. For example, said nucleic acid    can be produced from a nucleic acid amplification process, contained    in a cell or organism or otherwise be extracted from a cell or    organism. Non-limiting examples of nucleic acid amplification    processes that can be the source for the nucleic acid include, but    are not limited to, Polymerase Chain Reaction (PCR), Ligase Chain    Reaction (LCR), Strand Displacement Amplification (SDA),    Transcription-Mediated Amplification (TMA), Q-beta replicase    amplification (Q-beta) and Rolling Circle Amplification (RCA).-   d. As used herein, “polynucleobase strand” means a complete single    polymer strand comprising nucleobase subunits. For example, a single    nucleic acid strand of a double stranded nucleic acid is a    polynucleobase strand.-   e. As used herein, “nucleic acid” is a nucleobase    sequence-containing polymer, or polymer segment, having a backbone    formed from nucleotides, or analogs thereof. Preferred nucleic acids    are DNA and RNA. For the avoidance of any doubt, PNA is a nucleic    acid mimic and not a nucleic acid analog.-   f. As used herein, “peptide nucleic acid” or “PNA” means any    oligomer or polymer segment (e.g. oligomer block) comprising two or    more PNA subunits (residues), but not nucleic acid subunits (or    analogs thereof), including, but not limited to, any of the oligomer    or polymer segments referred to or claimed as peptide nucleic acids    in U.S. Pat. Nos. 5,539,082, 5,527,675, 5,623,049, 5,714,331,    5,718,262, 5,736,336, 5,773,571, 5,766,855, 5,786,461, 5,837,459,    5,891,625, 5,972,610, 5,986,053 and 6,107,470; all of which are    herein incorporated by reference. The term “peptide nucleic acid” or    “PNA” shall also apply to any oligomer or polymer segment comprising    two or more subunits of those nucleic acid mimics described in the    following publications: Lagriffoul et al., Bioorganic & Medicinal    Chemistry Letters, 4: 1081-1082 (1994); Petersen et al., Bioorganic    & Medicinal Chemistry Letters, 6: 793-796 (1996); Diderichsen et    al., Tett. Lett. 37: 475-478 (1996); Fujii et al., Bioorg. Med.    Chem. Lett. 7: 637-627 (1997); Jordan et al., Bioorg. Med. Chem.    Lett. 7: 687-690 (1997); Krotz et al., Tett. Lett. 36: 6941-6944    (1995); Lagriffoul et al., Bioorg. Med. Chem. Lett. 4: 1081-1082    (1994); Diederichsen, U., Bioorganic & Medicinal Chemistry Letters,    7: 1743-1746 (1997); Lowe et al., J. Chem. Soc. Perkin Trans.    1, (1997) 1: 539-546; Lowe et al., J. Chem. Soc. Perkin Trans. 11:    547-554 (1997); Lowe et al., J. Chem. Soc. Perkin Trans. 11:555-560    (1997); Howarth et al., J. Org. Chem. 62:5441-5450 (1997); Altmann,    K-H et al., Bioorganic & Medicinal Chemistry Letters, 7:1119-1122    (1997); Diederichsen, U., Bioorganic & Med. Chem. Lett., 8: 165-168    (1998); Diederichsen et al., Angew. Chem. Int. Ed., 37: 302-305    (1998); Cantin et al., Tett. Lett., 38: 4211-4214 (1997); Ciapetti    et al., Tetrahedron, 53: 1167-1176 (1997); Lagriffoule et al., Chem.    Eur. J., 3: 912-919 (1997); Kumar et al., Organic Letters 3(9):    1269-1272 (2001); and the Peptide-Based Nucleic Acid Mimics (PENAMs)    of Shah et al. as disclosed in WO96/04000. For the avoidance of    doubt, the linking of one or more amino acid subunits, or one or    more labels or linkers, to a PNA oligomer or segment (e.g. PNA    oligomer block) does not produce a PNA chimera.

In certain embodiments, a “peptide nucleic acid” or “PNA” is an oligomeror polymer segment comprising two or more covalently linked subunits ofthe formula:

wherein, each J is the same or different and is selected from the groupconsisting of H, R¹, OR¹, SR¹, NHR¹, NR¹ ₂, F, Cl, Br and I. Each K isthe same or different and is selected from the group consisting of O, S,NH and NR¹. Each R¹ is the same or different and is an alkyl grouphaving one to five carbon atoms that may optionally contain a heteroatomor a substituted or unsubstituted aryl group. Each A is selected fromthe group consisting of a single bond, a group of the formula;—(CJ₂)_(s)- and a group of the formula; —(CJ₂)_(s)C(O)—, wherein, J isdefined above and each s is a whole number from one to five. Each t is 1or 2 and each u is 1 or 2. Each L is the same or different and isindependently selected from: adenine, cytosine, guanine, thymine,uracil, 5-propynyl-uracil, 2-thio-5-propynyl-uracil, 5-methylcytosine,pseudoisocytosine, 2-thiouracil and 2-thiothymine, 2-aminopurine,N9-(2-amino-6-chloropurine), N9-(2,6-diaminopurine), hypoxanthine,N9-(7-deaza-guanine), N9-(7-deaza-8-aza-guanine) andN8-(7-deaza-8-aza-adenine), other naturally occurring nucleobase analogsor other non-naturally occurring nucleobases.

In certain other embodiments, a PNA subunit consists of a naturallyoccurring or non-naturally occurring nucleobase attached to theN-α-glycine nitrogen of the N-[2-(aminoethyl)]glycine backbone through amethylene carbonyl linkage; this currently being the most commonly usedform of a peptide nucleic acid subunit.

-   g. As used herein, “PNA chimera” means an oligomer or polymer    segment comprising two or more PNA subunits and one or more nucleic    acid subunits (i.e. DNA or RNA), or analogs thereof, that are    selected from different classes of subunits and that are linked by a    covalent bond but not a linker. For example, a PNA/DNA chimera would    comprise at least two PNA subunits covalently linked, via a chemical    bond, to at least one 2′-deoxyribonucleic acid subunit (For    exemplary methods and compositions related to PNA/DNA chimera    preparation See: WO96/40709). For purposes of this invention, a PNA    chimera includes a combination of different types of nucleobases    containing subunits (e.g. PNA, DNA, RNA) but the mere incorporation    of amino acid subunits, such as glycine, or one or more labels or    linkers, does not mean that an oligomer is PNA chimera.-   h. As used herein, “block”, “oligomer block” or “block oligomer”    means a peptide nucleic acid, PNA chimera, or PNA combination    oligomer that is designed and available to be ligated to a second    appropriately modified oligomer to thereby prepare a combination    oligomer or elongated combination oligomer, as appropriate. The    oligomer blocks may be unlabeled, labeled with one or more reporter    moieties and/or comprise one or more protected or unprotected    functional groups.-   i. As used herein “PNA combination oligomer” means a combination    oligomer comprising at least one PNA oligomer block or PNA chimera    oligomer block.-   j. As used herein, “combination oligomer” means an oligomer    comprising two or more oligomer blocks that are linked by a linker.-   k. As used herein, “linker” means a moiety of at least three atoms    in length that is not part a nucleobase containing backbone subunit    of the polymer wherein said at least three atoms covalently link the    nucleobase containing backbone subunits of two component oligomer    blocks.-   l. As used herein, “native oligomer” means a peptide nucleic acid,    nucleic acid or PNA chimera that does not comprise a linker that    separates two oligomer blocks. Thus, a native oligomer, even if a    chimera, comprises a backbone wherein the backbone subunits are    linked together either directly or though a bridging moiety of no    more than two atoms in length.-   m. As used herein, “gap” means a space, that is at least one    nucleobase in length, between the terminal nucleobases of two    oligomer blocks adjacently hybridized onto a target sequence (See    for example: FIG. 1).-   n. As used herein, the terms “label”, “reporter moiety” or    “detectable moiety” are interchangeable and refer to moieties that    can be attached to an oligomer block or combination oligomer, or    otherwise be used in a reporter system, to thereby render the    oligomer detectable by an instrument or method. For example, a label    can be any moiety that: (i) provides a detectable signal; (ii)    interacts with a second label to modify the detectable signal    provided by the first or second label; or (iii) confers a capture    function, i.e. hydrophobic affinity, antibody/antigen, ionic    complexation.-   o. As used herein, “sequence specifically” means hybridization by    base pairing through hydrogen bonding. Non-limiting examples of    standard base pairing includes adenine base pairing with thymine or    uracil and guanine base pairing with cytosine. Other non-limiting    examples of base-pairing motifs include, but are not limited to:    adenine base pairing with any of: 5-propynyl-uracil,    2-thio-5-propynyl-uracil, 2-thiouracil or 2-thiothymine; guanine    base pairing with any of: 5-methylcytosine or pseudoisocytosine;    cytosine base pairing with any of: hypoxanthine,    N9-(7-deaza-guanine) or N9-(7-deaza-8-aza-guanine); thymine or    uracil base pairing with any of: 2-aminopurine,    N9-(2-amino-6-chloropurine) or N9-(2,6-diaminopurine); and    N8-(7-deaza-8-aza-adenine), being a universal base, base pairing    with any other nucleobase, such as for example any of: adenine,    cytosine, guanine, thymine, uracil, 5-propynyl-uracil,    2-thio-5-propynyl-uracil, 5-methylcytosine, pseudoisocytosine,    2-thiouracil and 2-thiothymine, 2-aminopurine,    N9-(2-amino-6-chloropurine), N9-(2,6-diaminopurine), hypoxanthine,    N9-(7-deaza-guanine) or N9-(7-deaza-8-aza-guanine) (See: Seela et    al., Nucl. Acids, Res.: 28(17): 3224-3232 (2000)).-   p. As used herein, “condensation conditions” means conditions    suitable to condense/ligate two oligomer blocks in accordance with    the condensation/ligation chemistry chosen.-   q. As used herein “ligation” and “condensation” are interchangeable    and, because strictly speaking not all of the methods suitable for    linking the oligomer blocks are condensation reactions, refer to the    process of covalently linking two oligomer blocks to thereby form a    combination oligomer comprising a linker of at least three atoms in    length. It is also to be understood that the ligation/condensation    chemistry is not to be a limitation of these methods so long as it    produces a linker between the oligomer blocks. Non-limiting examples    of numerous ligation/condensation chemistries suitable for forming    combination oligomers are described herein with reference to FIGS.    28 b & 29 b-32.-   r. As used herein, “nucleobase protecting group” means a protecting    group covalently linked to a functional group of a nucleobase to    render the functional group unreactive during certain chemical    reactions (e.g. ligation/condensation). For example, the exocylic    amino groups of adenine, cytosine and guanine are typically    protected with a suitable protecting group during de novo chemical    oligomer synthesis. However, nucleobases need not be protected    during the ligation/condensation reactions described herein. For the    avoidance of doubt, formation of a salt of a functional group to    render the group unreactive during a chemical reaction is not a    nucleobase-protecting group, as used herein, since there is no    covalent link.-   s. As used herein, “quenching” means a decrease in fluorescence of a    fluorescent reporter moiety caused by energy transfer associated    with a quencher moiety, regardless of the mechanism.-   t. As used herein “solid support” or “solid carrier” means any solid    phase material upon which a combination oligomer is synthesized,    attached, ligated or otherwise immobilized. Solid support    encompasses terms such as “resin”, “solid phase”, “surface” and    “support”. A solid support may be composed of organic polymers such    as polystyrene, polyethylene, polypropylene, polyfluoroethylene,    polyethyleneoxy, and polyacrylamide, as well as co-polymers and    grafts thereof. A solid support may also be inorganic, such as    glass, silica, controlled-pore-glass (CPG), or reverse-phase silica.    The configuration of a solid support may be in the form of beads,    spheres, particles, granules, a gel, or a surface. Surfaces may be    planar, substantially planar, or non-planar. Solid supports may be    porous or non-porous, and may have swelling or non-swelling    characteristics. A solid support may be configured in the form of a    well, depression or other container, vessel feature or location. A    plurality of solid supports may be configured in an array at various    locations, addressable for robotic delivery of reagents, or by    detection means including scanning by laser illumination and    confocal or deflective light gathering.-   u. As used herein, “support bound” means immobilized on or to a    solid support. It is understood that immobilization can occur by any    means, including for example; by covalent attachment, by    electrostatic immobilization, by attachment through a ligand/ligand    interaction, by contact or by depositing on the surface.-   v. “Array” or “microarray” means a predetermined spatial arrangement    of oligomers present on a solid support or in an arrangement of    vessels. Certain array formats are referred to as a “chip” or    “biochip” (M. Schena, Ed. Microarray Biochip Technology,    BioTechnique Books, Eaton Publishing, Natick, Mass. (2000). An array    can comprise a low-density number of addressable locations, e.g. 2    to about 12, medium-density, e.g. about a hundred or more locations,    or a high-density number, e.g. a thousand or more. Typically, the    array format is a geometrically-regular shape that allows for    fabrication, handling, placement, stacking, reagent introduction,    detection, and storage. The array may be configured in a row and    column format, with regular spacing between each location.    Alternatively, the locations may be bundled, mixed, or homogeneously    blended for equalized treatment or sampling. An array may comprise a    plurality of addressable locations configured so that each location    is spatially addressable for high-throughput handling, robotic    delivery, masking, or sampling of reagents, or by detection means    including scanning by laser illumination and confocal or deflective    light gathering.    2. Description Of The Invention    I. General    PNA Synthesis:

Methods for the chemical assembly of PNAs are well known (See: U.S. Pat.Nos. 5,539,082, 5,527,675, 5,623,049, 5,714,331, 5,718,262, 5,736,336,5,773,571, 5,766,855, 5,786,461, 5,837,459, 5,891,625, 5,972,610,5,986,053 and 6,107,470; all of which are herein incorporated byreference (Also see: PerSeptive Biosystems Product Literature)). As ageneral reference for PNA synthesis methodology also please see: Nielsenet al., Peptide Nucleic Acids; Protocols and Applications, HorizonScientific Press, Norfolk England (1999).

Chemicals and instrumentation for the support bound automated chemicalassembly of peptide nucleic acids are now commercially available. Bothlabeled and unlabeled PNA oligomers are likewise available fromcommercial vendors of custom PNA oligomers. Chemical assembly of a PNAis analogous to solid phase peptide synthesis, wherein at each cycle ofassembly the oligomer possesses a reactive alkyl amino terminus that iscondensed with the next synthon to be added to the growing polymer.

PNA may be synthesized at any scale, from submicromole to millimole, ormore. Most conveniently, PNA is synthesized at the 2 μmole scale, usingFmoc/Bhoc, tBoc/Z, or MMT protecting group monomers on an ExpediteSynthesizer (Applied Biosystems) on XAL or PAL support; or on the Model433A Synthesizer (Applied Biosystems) with MBHA support; or on otherautomated synthesizers. Because standard peptide chemistry is utilized,natural and non-natural amino acids can be routinely incorporated into aPNA oligomer. Because a PNA is a polyamide, it has a C-terminus(carboxyl terminus) and an N-terminus (amino terminus). For the purposesof the design of a hybridization probe suitable for antiparallel bindingto the target sequence (the preferred orientation), the N-terminus ofthe probing nucleobase sequence of the PNA probe is the equivalent ofthe 5′-hydroxyl terminus of an equivalent DNA or RNA oligonucleotide.

When used in ligation reactions, the nature of the ligation chemistrychosen should be considered. For simplicity, we sometimes refer to oneof the oligomer blocks used in a ligation reaction as a terminal blockand the other as the condensation block. This distinction is generallyirrelevant except to distinguish between the different blocks especiallyif they contain the same nucleobase sequence. Often at least the natureof the functional groups that are used in the ligation will be differentfor the terminal and condensation blocks since they can be designed toaccommodate different ligation chemistries. However, when the oligomeris to be extended by multiple ligations, we will generally refer to theterminal oligomer block as the oligomer block produced from the firstligation or from the immediately preceding ligation step. Accordingly,depending on whether an oligomer block is a condensing oligomer block orterminal oligomer block may have an effect on the actual composition ofthe termini. Several non-limiting examples of ligation chemistries areillustrated in FIGS. 28-31 a & 31 b. Using no more than routineexperimentation as well as the description contained herein, one ofordinary skill in the art will easily be able to prepare combinationoligomers according to this invention.

The terminal blocks may comprise a C-terminal amide that is relativelyunreactive. In contrast, the condensing blocks may comprise a C-terminalend that is suitable for the ligation reaction. However, depending uponthe nature of the condensation chemistry, the C-terminal end of theoligomer may comprise a C-terminal acid or an alternative functionalgroup (See For Example: FIGS. 28 a-31 b). If a functional group, theterminus may or may not require the addition of a protecting groupdepending on the nature of the condensation/ligation chemistry. Sincethe oligomer blocks are themselves often prepared by de novo methods andbecause suitable commercial reagents and instrumentation are availablefor the production of PNA oligomers comprising either of a C-terminalamino acid, an alternative functional group or a C-terminal amide, oneof skill in the art can easily prepare the oligomer blocks of thedesired C-terminal configuration.

With respect to the N-terminus, again the exact configuration can dependon the nature of the ligation chemistry chosen and on whether or not theoligomer is a condensing oligomer block or a terminal oligomer block. Ifthe oligomer is a terminal oligomer block, the N-terminus may comprise areactive functional group whereas if the oligomer is a condensingoligomer block, the N-terminus can be capped. Non-limiting examples ofcapping include labeling the N-terminus with a label or otherwisereacting it with a relatively non-reactive moiety such as acetyl. If theN-terminus is to be involved in the ligation reaction, it will typicallyexist as a free amine unless alternative condensation chemistry isemployed (See For Example: FIGS. 28 a-31 b). Since the oligomer blocksare themselves prepared by de novo methods and because suitablecommercial reagents and instrumentation are available for the productionof PNA oligomers, one of skill in the art can easily prepare theoligomer blocks of the desired N-terminal configuration.

In addition to the modification of the termini for ligation, theoligomer blocks can be modified and/or properly protected to therebyincorporate functional groups for labeling or for attachment tosurfaces. Such functional groups can be utilized either before or afterligation depending upon factors such as: 1) the oligomer synthesischemistry (e.g. harsh deprotection conditions required that mightdestroy a label), the condensation/ligation chemistry chosen (e.g.functional groups of the desired label might interfere with thecondensation chemistry) and the intended use of the functional group(e.g. whether it is intended for labeling or for attachment to a solidsupport).

PNA Labeling/Modification:

Non-limiting methods for labeling PNAs are described in U.S. Pat. Nos.6,110,676, 6,280,964, WO99/22018, WO99/21881, WO99/37670 and WO99/49293,the examples section of this specification or are otherwise well knownin the art of PNA synthesis and peptide synthesis. Methods for labelingPNA are also discussed in Nielsen et al., Peptide Nucleic Acids;Protocols and Applications, Horizon Scientific Press, Norfolk, England(1999). Non-limiting methods for labeling the PNA oligomers that caneither be used as block oligomers or otherwise be used to preparecombination oligomers are as follows.

Because the synthetic chemistry of assembly is essentially the same, anymethod commonly used to label a peptide can often be adapted to effectthe labeling a PNA oligomer. Generally, the N-terminus of the polymercan be labeled by reaction with a moiety having a carboxylic acid groupor activated carboxylic acid group. One or more spacer moieties canoptionally be introduced between the labeling moiety and the nucleobasecontaining subunits of the oligomer. Generally, the spacer moiety can beincorporated prior to performing the labeling reaction. If desired, thespacer may be embedded within the label and thereby be incorporatedduring the labeling reaction.

Typically the C-terminal end of the polymer can be labeled by firstcondensing a labeled moiety or functional group moiety with the supportupon which the PNA oligomer is to be assembled. Next, the firstnucleobase containing synthon of the PNA oligomer can be condensed withthe labeled moiety or functional group moiety. Alternatively, one ormore spacer moieties (e.g. 8-amino-3,6-dioxaoctanoic acid; the“O-linker”) can be introduced between the label moiety or functionalgroup moiety and the first nucleobase subunit of the oligomer. Once themolecule to be prepared is completely assembled, labeled and/ormodified, it can be cleaved from the support deprotected and purifiedusing standard methodologies.

For example, the labeled moiety or functional group moiety can be alysine derivative wherein the ε-amino group is a protected orunprotected functional group or is otherwise modified with a reportermoiety. The reporter moiety could be a fluorophore such as5(6)-carboxyfluorescein or a quencher moiety such as4-((4-(dimethylamino)phenyl)azo)benzoic acid (dabcyl). Condensation ofthe lysine derivative with the synthesis support can be accomplishedusing standard condensation (peptide) chemistry. The α-amino group ofthe lysine derivative can then be deprotected and the nucleobasesequence assembly initiated by condensation of the first PNA synthonwith the α-amino group of the lysine amino acid. As discussed above, aspacer moiety may optionally be inserted between the lysine amino acidand the first PNA synthon by condensing a suitable spacer (e.g.Fmoc-8-amino-3,6-dioxaoctanoic acid) with the lysine amino acid prior tocondensation of the first PNA synthon.

Alternatively, a functional group on the assembled, or partiallyassembled, polymer can be introduced while the oligomer is still supportbound. The functional group will then be available for any purpose,including being used to either attached the oligomer to a support orotherwise be reacted with a reporter moiety, including being reactedpost-ligation (by post-ligation we mean at a point after the combinationoligomer has been fully formed by the performing of one or morecondensation/ligation reactions). This method, however, requires that anappropriately protected functional group be incorporated into theoligomer during assembly so that after assembly is completed, a reactivefunctional can be generated. Accordingly, the protected functional groupcan be attached to any position within the combination oligomer orblock, including, at the block oligomer termini, at a position internalto the oligomer blocks, or linked at a position integral to the linker.

For example, the ε-amino group of a lysine could be protected with a4-methyl-triphenylmethyl (Mtt), a 4-methoxy-triphenylmethyl (MMT) or a4,4′-dimethoxytriphenylmethyl (DMT) protecting group. The Mtt, MMT orDMT groups can be removed from the oligomer (assembled usingcommercially available Fmoc PNA monomers and polystyrene support havinga PAL linker; PerSeptive Biosystems, Inc., Framingham, Mass.) bytreatment of the synthesis resin under mildly acidic conditions.Consequently, a donor moiety, acceptor moiety or other reporter moiety,for example, can then be condensed with the ε-amino group of the lysineamino acid while the polymer is still support bound. After completeassembly and labeling, the polymer is then cleaved from the support,deprotected and purified using well-known methodologies.

By still another method, the reporter moiety is attached to thecombination oligomer or oligomer block after it is fully assembled andcleaved from the support. This method is preferable where the label isincompatible with the cleavage, deprotection or purification regimescommonly used to manufacture the oligomer. By this method, the PNAoligomer will generally be labeled in solution by the reaction of afunctional group on the polymer and a functional group on the label.Those of ordinary skill in the art will recognize that the compositionof the coupling solution will depend on the nature of oligomer andlabel, such as for example a donor or acceptor moiety. The solution maycomprise organic solvent, water or any combination thereof. Generally,the organic solvent will be a polar non-nucleophilic solvent. Nonlimiting examples of suitable organic solvents include acetonitrile(ACN), tetrahydrofuran, dioxane, methyl sulfoxide,N,N′-dimethylformamide (DMF) and N-methylpyrrolidone (NMP).

The functional group on the polymer to be labeled can be a nucleophile(e.g. an amino group) and the functional group on the label can be anelectrophile (e.g. a carboxylic acid or activated carboxylic acid). Itis however contemplated that this can be inverted such that thefunctional group on the polymer can be an electrophile (e.g. acarboxylic acid or activated carboxylic acid) and the functional groupon the label can be a nucleophile (e.g. an amino acid group).Non-limiting examples of activated carboxylic acid functional groupsinclude N-hydroxysuccinimidyl esters. In aqueous solutions, thecarboxylic acid group of either of the PNA or label (depending on thenature of the components chosen) can be activated with a water solublecarbodiimide. The reagent,1-ethyl-3-(3-dimethylamino-propyl)carbodiimide hydrochloride (EDC), is acommercially available reagent sold specifically for aqueous amideforming condensation reactions. Applicants have likewise observed thatsuch condensation reactions can be improved when1-Hydroxy-7-azabenzotriazole (HOAt) or 1-hydrozybenzotriazole (HOBt) ismixed with the EDC.

The pH of aqueous solutions can be modulated with a buffer during thecondensation reaction. For example, the pH during the condensation canbe in the range of 4-10. Generally, the basicity of non-aqueousreactions will be modulated by the addition of non-nucleophilic organicbases. Non-limiting examples of suitable bases includeN-methylmorpholine, triethylamine and N,N-diisopropylethylamine.Alternatively, the pH can be modulated using biological buffers such as(N-[2-hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid) (HEPES) or4-morpholineethane-sulfonic acid (MES) or inorganic buffers such assodium bicarbonate.

Chimera Synthesis and Labeling/Modification:

PNA chimeras are a combination of a nucleic acid and peptide nucleicacid subunits. Hence, the synthesis, labeling and modification of PNAchimeras can utilize methods known to those of skill in the art as wellas those described above. A suitable reference for the synthesis,labeling and modification of PNA chimeras can be found in WIPO publishedpatent application number WO96/40709, now issued as U.S. Pat. No.6,063,569, herein incorporated by reference. Moreover, the methodsdescribed above for PNA synthesis and labeling often can be use formodifying the PNA portion of a PNA chimera. Additionally, well-knownmethods for the synthesis and labeling of nucleic acids can often beused for modifying the nucleic acid portion of a PNA chimera. Exemplarymethods can be found in U.S. Pat. Nos. 5,476,925, 5,453,496, 5,446,137,5,419,966, 5,391,723, 5,391,667, 5,380,833, 5,348,868, 5,281,701,5,278,302, 5,262,530, 5,243,038, 5,218,103, 5,204,456, 5,204,455,5,198,540, 5,175,209, 5,164,491, 5,112,962, 5,071,974, 5,047,524,4,980,460, 4,923,901, 4,786,724, 4,725,677, 4,659,774, 4,500,707,4,458,066, and 4,415,732; all of which are herein incorporated byreference.

Labeled Combination Oligomers & Oligomer Blocks:

Whether a, peptide nucleic acid, PNA chimera, PNA combination oligomer,or variation thereof, the combination oligomers or oligomer blocks thatare used for the practice of this invention may be labeled with areporter moiety. Each label or reporter moiety can be linked to anyposition within the combination oligomer or oligomer block, including,at the block oligomer termini, at a position internal to the oligomerblocks, or linked at a position integral to the linker. Non-limitingexamples of reporter moieties (labels) suitable for directly labelingcombination oligomers or oligomer blocks used in the practice of thisinvention include: a quantum dot, a minor groove binder, a dextranconjugate, a branched nucleic acid detection system, a chromophore, afluorophore, a quencher, a spin label, a radioisotope, an enzyme, ahapten, an acridinium ester and a chemiluminescent compound. Quenchingmoieties are also considered labels. Other suitable labeling reagentsand preferred methods of attachment would be recognized by those ofordinary skill in the art of PNA, peptide or nucleic acid synthesis.Non-limiting examples are described or referred to above.

Non-limiting examples of haptens include 5(6)-carboxyfluorescein,2,4-dinitrophenyl, digoxigenin, and biotin.

Non-limiting examples of fluorochromes (fluorophores) include5(6)-carboxyfluorescein (Flu), 2′,4′,1,4,-tetrachlorofluorescein; and2′,4′,5′,7′,1,4-hexachlorofluorescein, other fluorescein dyes (See: U.S.Pat. Nos. 5,188,934; 6,008,379; 6,020,481, incorporated herein byreference), 6-((7-anino-4-methylcoumarin-3-acetyl)amino)hexanoic acid(Cou), 5(and 6)-carboxy-X-rhodamine (Rox), other rhodamine dyes (See:U.S. Pat. Nos. 5,366,860; 5,847,162; 5,936,087; 6,051,719; 6,191,278;6,248,884, incorporated herein by reference), benzophenoxazines (See:U.S. Pat. No. 6,140,500, incorporated herein by reference) Cyanine 2(Cy2) Dye, Cyanine 3 (Cy3) Dye, Cyanine 3.5 (Cy3.5) Dye, Cyanine 5 (Cy5)Dye, Cyanine 5.5 (Cy5.5) Dye Cyanine 7 (Cy7) Dye, Cyanine 9 (Cy9) Dye(Cyanine dyes 2, 3, 3.5, 5 and 5.5 are available as NHS esters fromAmersham, Arlington Heights, Ill.), other cyanine dyes (Kubista, WO97/45539), 6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein (JOE),5(6)-carboxy-tetramethyl rhodamine (Tamara), Dye 1 (FIG. 33 a), Dye2(FIG. 33 b) or the Alexa dye series (Molecular Probes, Eugene, Oreg.).

Non-limiting examples of enzymes include polymerases (e.g. Taqpolymerase, Klenow PNA polymerase, T7 DNA polymerase, Sequenase, DNApolymerase 1 and phi29 polymerase), alkaline phosphatase (AP),horseradish peroxidase (HRP), soy bean peroxidase (SBP)), ribonucleaseand protease.

Non-limiting examples of quenching moieties include diazo-containingmoieties such as aryldiazo compounds, e.g. dabcyl and dabsyl, homologscontaining one more additional diazo and/or aryl groups; e.g. FastBlack, (Nardone, U.S. Pat. No. 6,117,986), and substituted compoundswhere Z is a substituent such Cl, F, Br, C₁-C₆ alkyl, C₅-C₁₄ aryl,nitro, cyano, sulfonate, NR₂, —OR, and CO₂H, where each R isindependently H, C₁-C₆ alkyl or C₅-C₁₄ aryl according to the structures:

cyanine dyes (Lee, U.S. Pat. No. 6,080,868), including the exemplarystructure:

and other chromophores such as anthraquinone, malachite green,nitrothiazole, and nitroimidazole compounds and the like wherein thegroup X is the covalent attachment site of a bond or linker to thecombination oligomers of the invention.

A non-limiting example of a minor groove binder is CDPI₃, represented bythe structure:

where X are exemplary attachment sites to a combination oligomer(Dempcy, WO 01/31063).

Non-radioactive labeling methods, techniques, and reagents are reviewedin: Non-Radioactive Labeling, A Practical Introduction, Garman, A. J.Academic Press, San Diego, Calif. (1997)

Guidance in Label Choices when Ligating/Condensing Oligomer Blocks:

It will be apparent to one of skill in the art that when oligomer blocksare to be condensed/ligated in accordance with this invention, tothereby produce a combination oligomer, the entire nature of thepotentially reactive functional groups of the component oligomer blocksshould be considered for potential side or cross-reactions. Protectinggroups can also be used, as appropriate, to minimize or eliminatepotential side or cross-reactions. For example, when labeled oligomersare to be ligated, it is wise to consider the potential for reactivityof functional groups of the one or more labels in view of the nature ofthe various ligation chemistries that can be chosen.

By way of illustration, when performing condensation/ligation reactionsinvolving an amino group, carboxylic acid group and water solublecarbodiimide (e.g. Examples 2 & 6), the labels of the energy transferset (see discussion of energy transfer set below) should generally beselected to avoid unprotected reactive amino and carboxylic functionalgroups to thereby avoid possible side/cross reactions. As Examples 2 and6 demonstrate, it is possible to ligate labeled and unlabeled PNAoligomer blocks in good yield provided there are no functional groupsthat produce cross reactions during the condensation reaction.Consequently, one of skill in the art will therefore understand how toeffect optimal ligation/condensation conditions by consideration of thenature of the reactive functional groups of the component parts in viewof the nature of the particular ligation/condensation chemistry chosen.

Energy Transfer

For energy transfer to be useful in determining hybridization, thereshould be an energy transfer set comprising at least one energy transferdonor and at least one energy transfer acceptor moiety. Often, theenergy transfer set will include a single donor moiety and a singleacceptor moiety, but this is not a limitation. An energy transfer setmay contain more than one donor moiety and/or more than one acceptormoiety. The donor and acceptor moieties operate such that one or moreacceptor moieties accepts energy transferred from the one or more donormoieties or otherwise quenches the signal from the donor moiety ormoieties. Thus, in one embodiment, both the donor moiety(ies) andacceptor moiety(ies) are fluorophores. Though the previously listedfluorophores (with suitable spectral properties) might also operate asenergy transfer acceptors the acceptor moiety can also be a quenchermoiety such as 4-((-4-(dimethylamino)phenyl)azo) benzoic acid (dabcyl).The labels of the energy transfer set can be linked at the oligomerblock termini or linked at a site within the oligomer blocks. In oneembodiment, each of two labels of an energy transfer set can be linkedat the distal-most termini of the combination oligomer.

Transfer of energy between donor and acceptor moieties may occur throughany energy transfer process, such as through the collision of theclosely associated moieties of an energy transfer set(s) or through anon-radiative process such as fluorescence resonance energy transfer(FRET). For FRET to occur, transfer of energy between donor and acceptormoieties of a energy transfer set requires that the moieties be close inspace and that the emission spectrum of a donor(s) have substantialoverlap with the absorption spectrum of the acceptor(s) (See: Yaron etal. Analytical Biochemistry, 95: 228-235 (1979) and particularly page232, col. 1 through page 234, col. 1). Alternatively, collision mediated(radiationless) energy transfer may occur between very closelyassociated donor and acceptor moieties whether or not the emissionspectrum of a donor moiety(ies) has a substantial overlap with theabsorption spectrum of the acceptor moiety(ies) (See: Yaron et al.,Analytical Biochemistry, 95: 228-235 (1979) and particularly page 229,col. 1 through page 232, col. 1). This process is referred to asintramolecular collision since it is believed that quenching is causedby the direct contact of the donor and acceptor moieties (See: Yaron etal.). It is to be understood that any reference to energy transfer inthe instant application encompasses all of thesemechanistically-distinct phenomena. It is also to be understood thatenergy transfer can occur though more than one energy transfer processsimultaneously and that the change in detectable signal can be a measureof the activity of two or more energy transfer processes. Accordingly,the mechanism of energy transfer is not a limitation of this invention.

Detecting Energy Transfer in a Self-Indicating Combination Oligomer:

In certain embodiments, the combination oligomers are self-indicating.In one embodiment, a self-indicating combination oligomer can be labeledin a manner that is described in co-pending and commonly owned patentapplication U.S. Ser. No. 09/179,162 (now allowed), entitled: “Methods,Kits And Compositions Pertaining To Linear Beacons” and the related PCTapplication which has also now published as WO99/21881, both of whichare hereby incorporated by reference. These self-indicating combinationoligomers differ from those exemplary probes first described in U.S.Ser. No. 09/179,162 or WO99/21881 primarily in presence of the linkerthat is situated between two oligomer blocks to which one of either adonor or acceptor moiety is linked.

Hybrid formation between a self-indicating combination oligomer and atarget sequence can be monitored by measuring at least one physicalproperty of at least one member of the energy transfer set that isdetectably different when the hybridization complex is formed ascompared with when the combination oligomer exists in a non-hybridizedstate. We refer to this phenomenon as the self-indicating property ofthe combination oligomer. This change in detectable signal results fromthe change in efficiency of energy transfer between donor and acceptormoieties caused by hybridization of the combination oligomer to thetarget sequence.

For example, the means of detection can involve measuring fluorescenceof a donor or acceptor fluorophore of an energy transfer set. In oneembodiment, the energy transfer set may comprise at least one donorfluorophore and at least one acceptor (fluorescent or non-fluorescent)quencher such that the measure of fluorescence of the donor fluorophorecan be used to detect, identify or quantitate hybridization of thecombination oligomer to the target sequence. For example, there may be ameasurable increase in fluorescence of the donor fluorophore upon thehybridization of the combination oligomer to a target sequence (See:Examples 2, 3 and 5).

In another embodiment, the energy transfer set comprises at least onedonor fluorophore and at least one acceptor fluorophore such that themeasure of fluorescence of either, or both, of at least one donor moietyor one acceptor moiety can be used to can be used to detect, identify orquantitate hybridization of the combination oligomer to the targetsequence.

Detection of Energy Transfer in a Detection Complex:

In another embodiment, the combination oligomers of the presentinvention are labeled solely with a quencher moiety and are used as acomponent oligomer in a Detection Complex as more fully explained inU.S. Ser. No. 09/275,848, entitled: “Methods, Kits And CompositionsPertaining To Detection Complexes” and the related PCT application whichhas also now published as WO99/49293, both of which are hereinincorporated by reference. When the Detection Complex is formed, atleast one donor moiety of one component polymer is brought sufficientlyclose in space to at least one acceptor moiety of a second componentpolymer. Since the donor and acceptor moieties of the set are closelysituated in space, transfer of energy occurs between moieties of theenergy transfer set. When the Detection Complex dissociates, as forexample when a polymerase copies one of the strands of the DetectionComplex, the donor and acceptor moieties do not interact sufficiently tocause substantial transfer of energy from the donor and acceptormoieties of the energy transfer set and there is a correlating change indetectable signal from the donor and/or acceptor moieties of the energytransfer set. Consequently, Detection Complex formation/dissociation canbe determined by measuring at least one physical property of at leastone member of the energy transfer set that is detectably different whenthe complex is formed as compared with when the component polymers ofthe Detection Complex exist independently and unassociated.

Detectable and Independently Detectable Moieties/Multiplex Analysis:

In certain embodiments of this invention, a multiplex hybridizationassay is performed. In a multiplex assay, numerous conditions ofinterest are simultaneously or sequentially examined. Multiplex analysisrelies on the ability to sort sample components or the data associatedtherewith, during or after the assay is completed. In performing amultiplex assay, one or more distinct independently detectable moietiescan be used to label two or more different combination oligomers thatare to be used in an assay. By independently detectable we mean that itis possible to determine one label independently of, and in the presenceof, the other label. The ability to differentiate between and/orquantitate each of the independently detectable moieties provides themeans to multiplex a hybridization assay because the data correlateswith the hybridization of each of the distinct, independently labeledcombination oligomer to a particular target sequence sought to bedetected in the sample. Consequently, the multiplex assays of thisinvention can, for example, be used to simultaneously or sequentiallydetect the presence, absence, number, position or identity of two ormore target sequences in the same sample and in the same assay.

Example 5, below, is one example of a multiplex assay using theinvention discussed herein. In this Example, the each pair of probesused in the SNP analysis are labeled, each with a dye that fluoresces inan independently detectable color, such that: (i) if essentially onlyone of the two colors is detected, the sample is homozygous for one SNPcondition (ii) if essentially only the other of the two colors isdetected, the sample is homozygous for another SNP condition; but (iii)if both colors are detected the sample is heterozygous for the SNPcondition. In this way, all of the possible SNP permutations aredeterminable from one “multiplex” assay wherein the independentlydetectable color of the fluorescent dye labels linked to differentself-indicating combination oligomer probes used in the assay can beassociated with a particular condition of interest and the determinationof both labels in the same assay can be used to effectively call any oneof three different possible SNP conditions.

Spacer/Linker Moieties:

Generally, spacers can be used to minimize the adverse effects thatbulky labeling reagents might have on the hybridization properties ofprobes or primers. In the present invention, a linker can be used tolink two or more oligomer blocks of a combination oligomer. Non-limitingexamples of spacer/linker moieties suitable for use in this inventionconsist of: one or more aminoalkyl carboxylic acids (e.g. aminocaproicacid) the side chain of an amino acid (e.g. the side chain of lysine orornithine) one or more natural amino acids (e.g. glycine),aminooxyalkylacids (e.g. 8-amino-3,6-dioxaoctanoic acid), alkyl diacids(e.g. succinic acid), alkyloxy diacids (e.g. diglycolic acid) oralkyldiamines (e.g. 1,8-diamino-3,6-dioxaoctane). Spacer/linker moietiesmay also incidentally or intentionally be constructed to improve thewater solubility of the combination oligomer (For example see: Gildea etal., Tett. Lett. 39: 7255-7258 (1998)).

The linkers of this invention are abasic. By abasic we mean that they donot comprise a nucleobase. The linkers are at least three atoms inlength. For the avoidance of doubt, the atoms that define the linker arenot atoms that make up the monomer subunits of the oligomer. For examplethe 3′ and 5′ hydroxyl groups of a nucleotide subunit are not atoms ofthe linker. Similarly, the primary amine and carbonyl carbon of theN-(2-aminoethyl)-glycine moiety of a PNA subunit are not counted asbeing atoms of the linker.

For example, the linker can be one amino acid residue, two amino acidresidues, three amino acid residues, one E-linker residue, two E-Linkerresidues, one O-linker residue, two O-linker residues, one X-linkerresidue or two X-linker residues. More specifically, the linker can bethe amino acid glycine, the amino acid dimer gly-gly, the amino aciddimer gly-lys, the amino acid dimer lys-gly, the amino acid dimerglu-gly, the amino acid dimer gly-cys, the amino acid dimer cys-gly andthe amino acid dimer asp-gly.

Hybridization Conditions/Stringency:

Those of ordinary skill in the art of hybridization will recognize thatfactors commonly used to impose or control stringency of hybridizationinclude formamide concentration (or other chemical denaturant reagent),salt concentration (i.e., ionic strength), hybridization temperature,detergent concentration, pH and the presence or absence of chaotropes.Optimal stringency for a combination oligomer/target sequencecombination is often found by the well-known technique of fixing severalof the aforementioned stringency factors and then determining the effectof varying a single stringency factor. The same stringency factors canbe modulated to thereby control the stringency of hybridization of a PNAto a nucleic acid, except that the hybridization of a PNA is fairlyindependent of ionic strength. Optimal or suitable stringency for anassay may be experimentally determined by examination of each stringencyfactor until the desired degree of discrimination is achieved.

Suitable Hybridization Conditions:

Generally, the more closely related the background causing nucleic acidcontaminates are to the target sequence, the more carefully stringencymust be controlled. Blocking probes may also be used as a means toimprove discrimination beyond the limits possible by mere optimizationof stringency factors. Suitable hybridization conditions will thuscomprise conditions under which the desired degree of discrimination isachieved such that an assay generates an accurate (within the tolerancedesired for the assay) and reproducible result. Often this is achievedby adjusting stringency until sequence specific hybridization of theprobe and target sequence is achieved. Nevertheless, aided by no morethan routine experimentation and the disclosure provided herein, thoseof skill in the art will be able to determine suitable hybridizationconditions for performing assays utilizing the methods and compositionsdescribed herein.

Blocking Probes:

Blocking probes are nucleic acid or non-nucleic acid probes that can beused to suppress the binding of the probing nucleobase sequence of thecombination oligomer to a non-target sequence. Preferred blocking probesare PNA probes (See: Coull et al., U.S. Pat. No. 6,110,676, hereinincorporated by reference). The combination oligomers of this inventioncan likewise be used as blocking probes.

Typically, blocking probes are closely related to the probing nucleobasesequence and preferably they comprise one or more single point mutationsas compared with the target sequence sought to be detected in the assay.It is believed that blocking probes operate by hybridization to thenon-target sequence to thereby form a more thermodynamically stablecomplex than is formed by hybridization between the probing nucleobasesequence and the non-target sequence. Formation of the more stable andpreferred complex blocks formation of the less stable non-preferredcomplex between the probing nucleobase sequence and the non-targetsequence. Thus, blocking probes can be used with the methods andcompositions of this invention to suppress the binding of the nucleicacid or non-nucleic acid combination oligomer to a non-target sequencethat might be present in an assay and thereby interfere with theperformance of the assay. (See: Fiandaca et al. “PNA Blocker ProbesEnhance Specificity In Probe Assays”, Peptide Nucleic Acids: Protocolsand Applications, pp. 129-141, Horizon Scientific Press, Wymondham, UK,1999)

Probing Nucleobase Sequence:

The probing nucleobase sequence of a combination oligomer is thespecific sequence-recognition portion of the construct. We refer to theoligomers of this invention as combination oligomers, without regard tothe method of production, because their hybridization properties resultfrom the combined properties of the two or more component oligomerblocks, the nature of the linker and the opportunity for interactionbetween the blocks when the combination oligomer sequence specificallyhybridizes to a target sequence. Therefore, the probing nucleobasesequence of a combination oligomer is an aggregate nucleobase sequenceof the oligomer blocks that are designed to hybridize to a specifictarget sequence of contiguous nucleobases in a sample (See for Example:FIG. 1).Accordingly, the probing nucleobase sequence of the combinationoligomer is distributed (not necessarily evenly distributed) between atleast two oligomer blocks of the combination oligomer. Consequently,with due consideration to the requirements of a combination oligomer forthe assay format chosen, the length and sequence composition of theprobing nucleobase sequence of the combination oligomer will generallybe selected to form a double stranded complex with a target sequence ofcontiguous nucleobases (i.e. the oligomer blocks hybridize juxataposedsuch that there is no gap) under suitable hybridization conditions.

As discussed in Example 1, the combination oligomers of this inventionmay exhibit both a lower Tm than a native oligomer of equivalent probingnucleobase sequence and a greater ability to discriminate mismatcheswhen the mismatch is located other than at the termini of one oligomerblock. Hence, the design of the probing nucleobase sequence of acombination oligomer may involve the positioning of single pointmutations (mismatches, single nucleotide polymorphisms or SNPs) that areto be distinguished at a position other than at the termini of anoligomer block.

Non-limiting Examples of Ligation/Condensation Chemistries

With reference to FIGS. 28 a and 28 b, properly prepared oligomer blockscan be ligated using a carbodiimide, such as the water-solublecarbodiimide 1-Ethyl-3-(3-Dimethylamino-propyl) carbodiimidehydrochloride (EDC). As illustrated, typically one of the oligomerblocks comprises a carboxylic acid moiety and the other comprises anamine group. As PNA oligomers, whether or not they comprise linkednatural amino acid moieties, can comprise an amine terminus and acarboxylic acid terminus, it is an advantage that PNA oligomer blocks donot require modification to facilitate this ligation chemistry. Theoligomers can be ligated in an aqueous solution, optionally containing 1percent to 75 percent organic modifier (v/v). The pH can be less than6.5. Applicants have observed that the addition of an activating reagentsuch as a triazole compound (e.g. 1-Hydroxy-7-azabenzotriazole (HOAt) or1-Hydroxybenzotriazole (HOBt)) will increase the overall yield of thecondensation/ligation reaction. Accordingly it is recommended that anactivation reagent be used with the carbodiimide to effect the ligationwhen this chemistry is chosen.

Again with reference to FIGS. 28 a and 28 b, the product of ligation ofFIG. 28 a is a native PNA oligomer whereas the product of ligation ofFIG. 28 b is a combination oligomer comprising a gly-gly linker.Accordingly, it is to be understood that oligomer blocks can be selectedto prepare both native and combination oligomers and that the choice ofoligomer blocks of, for example, a library can be produced with thedesired product of condensation/ligation in mind.

With reference to FIGS. 29 a, 29 b, 29 c, 30 a and 30 b, several optionsfor the ligation/condensation of oligomer blocks are illustrated whereinsodium cyanoborohydride is used as reducing reagent. It is to beunderstood that sodium cyanoborohydride is one of many reducing reagentsthat could be used to effect the ligation of the oligomer blocks usingthese strategies for ligation.

With reference to FIGS. 29 a and 29 b, one of the oligomer blocks to beligated comprises an amine and the other oligomer block to be ligatedcomprises an aldehyde. The oligomer blocks can be brought into contactto thereby form an imine. Because imine formation is reversible, theimine is often reduced, by for example sodium cyanoborohydride, tothereby form the ligated combination oligomer. With reference to FIGS.29 a and 29 b, the illustrations are analogous to that observed withFIGS. 28 a and 28 b, respectively in that the product of ligation iseither a native oligomer or a combination oligomer, in this casecomprising an N-[2-aminoethyl]-glycine linker.

With reference to FIGS. 29 c, 30 a and 30 b, one of the oligomer blocksto be ligated is an aldehyde or ketone, such as glycinal or β-alinal,and the other oligomer block to be ligated comprises anaminooxy-containing moiety such as aminooxyacetyl. Reaction of properlymodified oligomer blocks results in the formation of an iminoxycombination oligomer that is more stable than an imine. Accordingly, theiminoxy combination oligomer can be used as prepared or can optionallybe reduced with, for example, sodium cyanoborohydride to thereby a morestable combination oligomer comprising a linker, as illustrated.

With reference to FIGS. 31 a, 31 b and 31 c, in each case one of theoligomer blocks comprises a nucleophilic thiol and a leaving group. FIG.31 a illustrates ligation in accordance with Lu et al. (J. Am. Chem.Soc., 118(36): 8518-8523 (1996)) to thereby form a combination oligomer.Reaction of a nucleophilic thiol, such as 2-aminoethly thiol (FIG. 31c), 2-thioacetyl or 3-thiopropionyl, with, for example, eitherhaloacetyl (FIG. 31 b), malimido (FIG. 31 c) or vinyl will likewiseproduce a combination oligomer.

Immobilization of Combination Oligomers To A Solid Support Or Surface:

One or more of the combination oligomers of this invention mayoptionally be immobilized to a surface or solid support for thedetection of a target sequence. Immobilization can, for example, be usedin capture assays and to prepare arrays.

The block oligomers and/or combination oligomers can be immobilized to asurface using the well known process of UV-crosslinking. The oligomerblocks can also be synthesized on the surface in a manner suitable fordeprotection but not cleavage from the synthesis support (See: Weiler,J. et al, Hybridization based DNA screening on peptide nucleic acid(PNA) oligomer arrays, Nucl. Acids Res., 25, 14:2792-2799 (July 1997)).In still another embodiment, one or more combination oligomers can becovalently linked to a surface by the reaction of a suitable functionalgroup on the oligomer with a functional group of the surface (See:Lester, A. et al, “PNA Array Technology”: Presented at BiochipTechnologies Conference in Annapolis (October 1997)). This method isadvantageous as compared to several of the other methods since theoligomers deposited on the surface for immobilization can be highlypurified and attached using a defined chemistry, thereby possiblyminimizing or eliminating non-specific interactions.

Methods for the chemical attachment of oligomer blocks or combinationoligomers to surfaces may involve the reaction of a nucleophilic group,(e.g. an amine or thiol) of the probe to be immobilized, with anelectrophilic group on the support to be modified. Alternatively, thenucleophile can be present on the support and the electrophile (e.g.activated carboxylic acid) present on the oligomer. Because native PNApossesses an amino terminus, a PNA may or may not require modificationto thereby immobilize it to a surface (See: Lester et al., Posterentitled “PNA Array Technology”).

Conditions suitable for the immobilization of a combination oligomer toa surface will generally be similar to those conditions suitable for thelabeling of the polymer. The immobilization reaction is essentially theequivalent of labeling whereby the label is substituted with the surfaceto which the polymer is to be linked (see above).

Numerous types of solid supports derivatized with amino groups,carboxylic acid groups, isocyantes, isothiocyanates and malimide groupsare commercially available. Non-limiting examples of suitable solidsupports include membranes, glass, controlled pore glass, polystyreneparticles (beads), silica and gold nanoparticles. All of the aboverecited methods of immobilization are not intended to be limiting in anyway but are merely provided by way of illustration.

Non-Limiting List of Advantages Associated with the Present Invention:

Combination oligomers comprising unprotected nucleobases can beefficiently ligated in the absence of a template. The efficiency ofligation also does not appear to be largely dependent upon scale therebyfacilitating a broad range of use, particularly for numerousapplications where the cost of conventional de novo oligomer synthesisis prohibitive such as where the amount of oligomer generated can be farin excess of that which is required. Hence, the present method enablesthe production of oligomers of desired nucleobase sequence in desiredquantities and can possibly produce substantial cost savings as comparedwith de novo methods.

Combination oligomers of desired nucleobase sequence can be rapidly andefficiently prepared in a single step from a readily available libraryof oligomer blocks (the “library approach”). The ability to rapidlymanufacture oligomers of defined nucleobase sequence that can bind to atarget sequence is enabling to high throughput applications such asnucleic acid sequencing, SNP analysis, genetic analysis, expressionanalysis and array production because tens, hundreds, thousands, tens oreven hundreds of thousands and possibly millions of probes can beproduced in a very time critical manner.

Combination oligomers, although generally exhibiting a lower absolute Tmas compared with native oligomers, may exhibit a larger ΔTm when the twooligomer blocks hybridize juxtaposed to a binding pair such that thereis no gap (See: FIG. 1). Because of this larger ΔTm, as compared withthe native oligomer, these combination oligomers can be more specificand discriminating as compared with the native oligomers (See: Egholm etal., Nature, 365: 566-568 (1993) wherein it is noted that the larger ΔTmfor a PNA mismatch as compared with that of a nucleic acid mismatch is ameasure of the discriminating power of the molecule in a hybridizationreaction). Because of the high discriminating power of the combinationoligomers, an assay for a single point mutation (also know as a singlenucleotide polymorphism (SNP)) can be designed wherein the oligomeroperates in essentially a binary mode such that it is either bound orunbound depending upon whether or not the wild type or mutant target ispresent. The hybrid of a 10-mer PNA combination oligomer and itscomplementary target sequence typically exhibits a Tm that is slightlyabove ambient temperature (e.g. 35-45° C.). However, one or morenon-complementary nucleobases in the hybrid will lower the Tm below roomtemperature. Hence, 10-mer PNA combination oligomers can be particularlyuseful and discriminating in analyses that are performed at ambienttemperature. Since analysis at ambient temperature avoids the need forexpensive temperature control equipment, this property of the hybridsand assays of this invention convey a substantial advantage to theordinary practitioner.

Combination oligomers can be designed to comprise an enzyme cleavagesite as the linker that links two oligomer blocks. The incorporation ofsuch a cleavage site can be advantageous when considered in combinationwith Applicant's observation that the combination oligomers can,depending upon the nature of the enzyme and cleavage site present, beprotected from cleavage as a result of the binding of the combinationoligomer to a suitable binding pair (e.g. a target sequence). Thus, byincorporating such a linker, it is an advantage that certain combinationoligomers can be used as substrates in an assay to, among other uses,determine whether or not a potential binding partner binds to thecombination oligomer since whether or not there is binding, and in whatamount, can be used to determine whether or not, and in what amount, thebinding partner (e.g. a target sequence) is present in a sample.

Oligomer purification by conventional methods such a high performanceliquid chromatography (HPLC) can expensive and time consuming. Becausethe combination oligomers are themselves produced from purifiedsubunits, delivery can be more rapid and cost effective where theproduct need not be purified post ligation. Moreover, because theunreacted component oligomer blocks that are ligated are typically tooshort to form a stable hybrid at or above ambient temperature, andtherefore are not necessarily problematic impurities, in manyapplications there may not be a requirement that the combinationoligomer be purified post-ligation and prior to use. This can facilitatethe rapid availability of probes/primers of desired composition once aproper library has been constructed.

Non-Limiting Examples of Applications for the Present Invention:

The method, kits, compositions, libraries and arrays of this inventionare useful in many areas or applications of scientific investigation.For example, this invention may be useful for the detection,identification and/or enumeration of viruses, bacteria and eucarya (e.g.pathogens) in food, beverages, water, pharmaceutical products, personalcare products, dairy products or in samples of plant, animal, human orenvironmental origin. The invention may also be useful for the analysisof raw materials, equipment, products or processes used to manufactureor store food, beverages, water, pharmaceutical products, personal careproducts dairy products or environmental samples. Additionally, thisinvention may be useful for the detection of bacteria and eucarya (e.g.pathogens) in clinical specimens, equipment, fixtures or products usedto treat humans or animals as well as in clinical samples and clinicalenvironments. For example, the analysis for microorganisms of interestcan be performed using PNA-FISH or multiplex PNA-FISH (See: Example 3 ofthis specification as well as co-pending and co-owned U.S. applicationSer. Nos. 09/335,629 and 09/368,089).

The method, kits, compositions, libraries and arrays of this inventionare particularly useful in areas such as expression analysis, singlenucleotide polymorphism (SNP) analysis (See: Example 5, below), geneticanalysis of humans, animals, fungi, yeast, viruses, and plants(including genetically modified organisms), therapy monitoring,pharmacogenomics, pharmacogenetics, epigenomics, and high throughputscreening operations. The libraries of this invention may be useful forthese probe intensive applications because they facilitate the massive,rapid, efficient and appropriately scaled synthesis of highlyselective/discriminating combination oligomers; a requirement that hasyet to be adequately fulfilled to thereby fully enable these probe orprimer intensive applications.

II. Exemplary Embodiments of the Invention

Combination Oligomers in General:

This invention pertains to the field of combination oligomers, includingthe block synthesis of combination oligomers in the absence of atemplate, as well as related methods, kits, libraries and othercompositions. The combination oligomers of this invention can beproduced either by stepwise assembly of monomers/synthons (de novosynthesis) using commercially available chemicals and instrumentation.Alternatively, the combination oligomers can be produced by ligation asgenerally described below (the “library approach”); for example asspecifically described in Examples 2 and/or Example 6 of thespecification. One of skill in the art can determine the mode ofcombination oligomer production that is best suited for the applicationchosen in view of one's own resources. Consequently, it is to beunderstood that the method of production of a combination oligomer isnot intended to be a limitation of the present invention.

As used herein a combination oligomer is a composition comprising two ormore oligomer blocks independently selected from peptide nucleic acid,PNA chimera and PNA combination oligomer, whether or not labeled,wherein said oligomer blocks are linked by a linker. This linker canoptionally be a cleavage site for an enzyme. The aforementionedcomposition is referred to herein as a combination oligomer, withoutregard to its method of production, because its hybridization propertiesresult from the combined properties of the two component oligomer blocksas well as the nature of the linker and the opportunity for interactionbetween the blocks when the combination oligomer hybridizes to a targetsequence.

It is to be understood that combination oligomers can be used as probesor primers in numerous applications. The requirement for probes ismerely that they hybridize to a target sequence with sequencespecificity. Thus, when used as a probe, there are no additionallimitations on specific features of the combination oligomer. However,when used as a primer, it is a requirement that the combination oligomercontain moieties suitable for the recognition and operation of an enzymesince polymerase enzymes are not known to operate on unmodified PNAoligomers (See: Lutz et al., J. Am. Chem. Soc. 119: 3177-3178 (1997)).

It is to be understood that for many embodiments of this invention, itis this generic embodiment of a combination oligomer that is to beconsidered with respect to the description of the invention. Unlessotherwise specifically noted herein, the novel combination oligomersreferred to herein as self-indicating combination oligomers andsubstrate combination oligomers may comprise limitations not generallyapplicable to other embodiments of the invention.

Novel Combination Oligomers:

In one embodiment, this invention pertains to novel combinationoligomers. The novel combination oligomers of this invention include,but are not necessarily limited to, substrate combination oligomers orself-indicating combination oligomers, as discussed below. (See also:Examples 1, 4 & 5, below). Whether a substrate combination oligomer orself-indicating combination oligomer, these novel combination oligomerscomprise a segment of the formula: A-W-C or A-B-C, wherein W or B areused to distinguish whether or not, respectively, the linker comprisesan enzyme cleavage site. Thus, the novel substrate combination oligomersof this invention comprise the linker W that is a substrate for anenzyme whereas the self-indicating combination oligomers are generallyillustrated by use of the linker B. However, a self-indicatingcombination oligomer may also be a substrate combination oligomer (Seefor example: Example 4).

According to the invention, A and C are oligomer blocks independentlyselected either as peptide nucleic acid, PNA chimera or PNA combinationoligomer. The linker B or W, as the case may be, is a linkage that is atleast three atoms in length that covalently links oligomer block A tooligomer block C. In this configuration, one or more additional oligomerblocks or other moieties such as a solid support, one or more protectedor unprotected functional groups or one or more labels may be covalentlyattached to either or both of A and C.

Self-Indicating Combination Oligomers:

In one embodiment, this invention pertains to self-indicatingcombination oligomers. Accordingly, this invention also pertains to acomposition of covalently linked oligomer blocks comprising a segment ofthe formula: A-B-C. Oligomer blocks A and C are each independently apeptide nucleic acid, PNA chimera or PNA combination oligomer and areoptionally linked to other moieties of the segment. The linker B is atleast three atoms in length and covalently links oligomer block A tooligomer block C. Moreover, oligomer blocks A and C taken togetherencode a probing nucleobase sequence that is designed to sequencespecifically hybridize to a target sequence of contiguous nucleobases tothereby form a double stranded target sequence/combination oligomercomplex.

A self-indicating combination oligomer further comprises an energytransfer set of labels such that at least one acceptor moiety of theenergy transfer set is linked to one of the linked oligomer blocks ofthe composition whilst at least one donor moiety of the energy transferset is linked to another of the linked oligomer blocks of thecomposition wherein labels of the set are linked to the combinationoligomer at positions that facilitate a change in detectable signal ofat least one label when the combination oligomer is hybridized to atarget sequence as compared to when the combination oligomer is in anon-hybridized state. As previously discussed, self-indicatingcombination oligomers are so named because there occurs, through anyenergy transfer mechanism, a change in detectable signal uponhybridization of the oligomer as compared to when the oligomer exists ina non-hybridized state. Accordingly, the state of hybridization of theoligomer can be determined in either real-time or at the end point of anassay. Moreover sets of independently detectable combination oligomerscan be prepared for multiplex analysis as demonstrated in Example 5.

In other embodiments of this invention, the self-indicating combinationoligomer may optionally further comprises one or more protected orunprotected functional groups or is otherwise labeled with one or moreadditional reporter moieties. According to the invention, the functionalgroups or reporter moieties can, each independently, be linked at theoligomer block termini, linked at a position internal to the oligomerblocks or linked at a position integral to the linker. Suitable reportermoieties have been previously described herein. The one or moreprotected or unprotected functional groups can be used, whendeprotected, to link a reporter moiety thereto or otherwise be used tolink the combination oligomer to a support. Consequently, theself-indicating combination oligomers can be support bound, including asappropriate, being one oligomer of an array of oligomers, including anarray of self-indicating combination oligomers.

Substrate Combination Oligomers:

In another embodiment, this invention pertains to substrate combinationoligomers. Accordingly, this invention also pertains to a composition ofcovalently linked oligomer blocks comprising a segment of the formula:A-W-C. Oligomer blocks A and C are each independently a peptide nucleicacid, PNA chimera or PNA combination oligomer. The linker W is at leastthree atoms in length and covalently links block A to block C. Thelinker W also comprises is a cleavage site for an enzyme.

Non-limiting examples of the linker W that comprises a cleavage sitesinclude, but are not limited to: lys-X, arg-X, Glu-X, asp-X, asn-X,phe-X, leu-X, lys-gly, arg-gly, glu-gly and asp-glu, wherein X is anynaturally occurring amino acid. A list of non-limiting examples ofenzymes suitable for cleaving one or more of these substrates include:Endoprotinase Glu-C (EC 3.4.21.19), Lys-C (EC 3.4.21.50), Arg-C (EC3.4.22.8), Asp-N (EC 3.4.24.33), Papain (EC 3.4.22.2), Pepsin (EC3.4.23.1), Proteinase K (3.4.21.14), chymotrypsin (EC 3.4.21.1) andtrypsin (3.4.21.4).

In other embodiments of this invention, the substrate combinationoligomer may optionally further comprises one or more protected orunprotected functional groups or is otherwise labeled with one or morereporter moieties. According to the invention, the functional groups orreporter moieties can, each independently, be linked at the oligomerblock termini, linked at a position internal to the oligomer blocks orlinked at a position integral to the linker. Suitable reporter moietieshave been previously described herein. The one or more protected orunprotected functional groups can be used, when deprotected, to link areporter moiety thereto or otherwise be used to link the combinationoligomer to a support. Consequently, the substrate combination oligomer,whether or not it is labeled, can be support bound, including asappropriate, being one oligomer of an array of oligomers, including anarray of substrate combination oligomers.

Hybrid of a Target Sequence and a Combination Oligomer:

In another embodiment, this invention pertains to a compositioncomprising a polynucleobase strand and a combination oligomer sequencespecifically hybridized to a target sequence of contiguous nucleobaseswithin the polynucleobase strand to thereby form a double strandedtarget sequence/combination oligomer complex (See FIG. 1; Also note thatthis configuration is sometimes referred to herein as being hybridizedjuxtaposed to the target sequence such that there is no gap or gapbase). The combination oligomer comprises a first and a second oligomerblock that are each independently a peptide nucleic acid, PNA chimera orPNA combination oligomer. The first and second oligomer blocks arecovalently linked by a linker of at least three atoms in length. Thehybrid can be formed by contacting the combination oligomer with thepolynucleobase strand under suitable hybridization conditions.

The combination oligomer of the hybrid may be unlabeled or furthercomprise protected or unprotected functional groups and/or reportermoieties. The combination oligomer may be a self-indicating combinationoligomer. The linker of the combination oligomer may comprise a cleavagesite for an enzyme. The hybrid may exist free in solution or be supportbound. In certain embodiments, the hybrid may exist at a unique positionof an array.

Method For Determining A Target Sequence

In yet another embodiment, this invention pertains to a method fordetermining a target sequence of contiguous nucleobases. The methodcomprises contacting the target sequence with a combination oligomer,under suitable hybridization conditions, wherein the combinationoligomer comprises a first oligomer block and a second oligomer blockthat are each independently a peptide nucleic acid, PNA chimera or PNAcombination oligomer. The first and second oligomer blocks are linkedcovalently to each other by a linker that is at least three atoms inlength. Moreover, the first and second oligomer blocks can sequencespecifically hybridize to the target sequence of contiguous nucleobasesto thereby form a double stranded target sequence/combination oligomercomplex. Accordingly, the aggregate of the first and second oligomerblocks comprise the probing nucleobase sequence. Complex formation isdetermined to thereby determine the target sequence since the complexdoes not form in the absence of the target sequence. Determination ofthe complex includes, but is not limited to, determining the presence,absence, quantity (amount) or position of the complex to therebydetermine the presence, absence, quantity (amount), position or identityof the target sequence (See for example: Examples 3-5).

According to the method, the combination oligomer of the hybrid may beunlabeled or further comprise protected or unprotected functional groupsand/or reporter moieties. The combination oligomer may be aself-indicating combination oligomer. The linker of the combinationoligomer may comprise a cleavage site for an enzyme. The complex mayexist free in solution or be support bound. In certain embodiments, thecomplex may exist at a unique position of an array.

It is to be understood that where the combination oligomer is labeled,determination of complex formation may involve the determination of oneor more of the labels. However, where the combination oligomer isunlabeled, the target sequence, or polynucleobase strand comprising thetarget sequence, can optionally be labeled such that determination ofthe label facilitates the determination of the formation of the complex.In still another embodiment, neither of the combination oligomer or thetarget sequence is labeled. Even though the complex is not directlylabeled, it is still possible to determine complex formation. Forexample, since the complex comprises at least a segment of a PNA/nucleicacid hybrid, the hybrid can be determined using an antibody that hasbeen raised to determine PNA/nucleic acid hybrids (See: Hyldig-Nielsenet al., U.S. Pat. No. 5,612,458, herein incorporated by reference).

Method for Determining the Zygosity of a SNP

In still another embodiment, this invention pertains to a method fordetermining the zygosity of a nucleic acid for a single nucleotidepolymorphism (SNP). The method comprises contacting a nucleic acidsample with at least two independently detectable combination oligomers.Each independently detectable combination oligomer comprises a firstoligomer block and a second oligomer block that are each independently apeptide nucleic acid, PNA chimera or PNA combination oligomer. Theoligomer blocks are linked covalently to each other by a linker that isat least three atoms in length. The first and second oligomer blockstaken together encode a probing nucleobase sequence that is designed tosequence specifically hybridize to a target sequence of contiguousnucleobases in a polynucleobase strand of the nucleic acid sample, ifpresent, to thereby form a double stranded target sequence/independentlydetectable combination oligomer complex. The probing nucleobase sequencein each independently detectable combination oligomer differs from theother by at least one nucleobase (the SNP nucleobase). However, theprobing nucleobase sequence may differ by more than one nucleobase,depending on probe design, provided however that they differ by a singlenucleobase at the SNP to be determined (See for example: Example 5,Table 9 and particularly the sets of PNA combination oligomer probes forSNPs 6876 and 6879).

According to the method, the nucleic acid sample and combinationoligomers are contacted with one or more reagents suitable forperforming a nucleic amplification reaction that amplifies the nucleicacid present in the sample and the nucleic acid amplification isperformed in the presence of the nucleic acid, the combination oligomersand the reagents. Non-limiting examples of nucleic acid amplificationreactions include: Polymerase Chain Reaction (PCR), Ligase ChainReaction (LCR), Strand Displacement Amplification (SDA),Transcription-Mediated Amplification (TMA), Q-beta replicaseamplification (Q-beta) and Rolling Circle Amplification (RCA).

Complex formation for each independently detectable combinationoligomer/target sequence complex is determined to thereby determinewhether the nucleic acid is heterozygous or homozygous for a particularSNP. Complex determination can be correlated with the zygosity state ofa particular SNP, since the complexes will not form in the absence ofthe respective target sequence of contiguous nucleobases for eachparticular combination oligomer. Moreover, signal for the twoindependently detectable combination oligomers provides all of theinformation needed to determine the three possible genotype statesdepending on which complexes do and do not form.

In one embodiment, the independently detectable combination oligomersare independently detectable, self-indicating combination oligomers.According to the method a determination, under suitable hybridizationconditions, is made of any change in detectable signal arising from atleast one of the labels of each of the independently detectable energytransfer sets as a measure of whether or not each of the combinationoligomers is hybridized to their respective target sequence ofcontiguous nucleobases. Such determination can be performed eitherduring the process of the nucleic acid amplification (e.g. in real-time)or after the nucleic acid amplification reaction is completed (e.g. atthe end-point). According to the method, the result of the change insignal for at least one label of each energy transfer set of eachcombination oligomer is correlated with a determination of the formationof each of the two possible target sequence/independently detectableself-indicating combination oligomer complexes. Based upon this data,one of the three possible states of zygosity of the sample for aparticular SNP can be determined (See for example: Example 5). Thetarget sequence/combination oligomer complex/hybrid can be free insolution or it can be support bound when the determination is made.

Non-Template Directed Methods for Forming Combination Oligomers

In another embodiment, this invention is directed to a method forforming a combination oligomer from oligomer blocks. The methodcomprises reacting a first oligomer block, a second oligomer block, andoptionally a condensation reagent or reagents under condensationconditions to thereby form a combination oligomer having a linker of atleast three atoms in length that covalently links the first oligomerblock to the second oligomer block. According to the method, the firstand second oligomer blocks are each independently a peptide nucleicacid, PNA chimera or PNA combination oligomer. Neither of the first orsecond oligomer blocks is support bound and the combination oligomerforms in the absence of a template. The ligation/condensation reactioncan be performed in aqueous solution. The nucleobases need not beprotected during the condensation/ligation reaction.

In yet another embodiment, this invention pertains to another method forforming combination oligomers from oligomer blocks. The method comprisesreacting a first oligomer block, a second oligomer block, and optionallya condensation reagent or reagents under condensation conditions tothereby form a combination oligomer having a linker of at least threeatoms in length that covalently links the first oligomer block to thesecond oligomer block. The first and second oligomer blocks are eachindependently a peptide nucleic acid, PNA chimera or PNA combinationoligomer. The nucleobases of the oligomer blocks do not compriseprotecting groups and the combination oligomer forms in the absence of atemplate. The ligation/condensation can be performed in aqueoussolution. Moreover, one of the oligomer blocks may or may not be supportbound.

Regardless of the method of forming a combination oligomer as describedabove, in another embodiment, the product of the condensation/ligationreaction can optionally be further lengthened/elongated. Hence, thecombination oligomer, as formed, can be used as an oligomer block suchthat repeating the method produces a further lengthened/elongatedoligomer. According to the method, the combination oligomer, aspreviously formed, can optionally be deprotected, as may be required, tofacilitate the next condensation/ligation step. The combinationoligomer, as previously formed and optionally deprotected, is reactedwith a third oligomer block and optionally a condensation reagent orreagents under condensation conditions. This forms the elongatedcombination oligomer having a covalent linkage of at least three atomsin length that covalently links the third oligomer block to thecombination oligomer wherein, the elongated combination oligomer formsin the absence of a template. In accordance with this method, thisprocess can be optionally repeated until the combination oligomer is ofthe desired length. Such a process of continued elongation can, forexample, be useful for the preparation of arrays since longer oligomersare often used for this application.

According to this method of the invention, the component oligomer blocksare either fully unprotected or one or both are partially protected.Accordingly, the oligomer blocks may comprise protected functionalgroups. By partially protected we mean that an electrophilic ornucleophilic functional group of the oligomer blocks are protected fromreaction, during the condensation, by a removable protecting group; itbeing self evident that the protecting group can be removed, forexample, after the condensation reaction is performed to thereby preparethe combination oligomer for subsequence condensation/ligation, tointroduce a label or to link the combination oligomer to a support.

Despite the absence of a template, Applicants have been able to rapidly,efficiently and repeatedly obtain combination oligomers in greater thatfifty percent (50%) yield (See: Examples 2 and 6), whether or not theoligomer blocks are labeled. The efficiency of this non-templatedirected ligation is surprising in view of published results wherein itis noted that essentially no ligation was observed in the absence of atemplate (See: Koppitz et al., J. Am. Chem. Soc. 120: 4563-4569 (1998)at page 4565, col. 1, lines 16-20 as well as Farèse et al., Tett. Lett.37: 1413-1416 (1996)).

The ligation/condensation methods of this invention can be used for theproduction of both labeled and unlabeled combination oligomers,including without limitation, both substrate combination oligomers aswell as self-indicating combination oligomers. The combination oligomersproduced by the methods of this invention may otherwise comprise one ormore protected or unprotected functional groups (the presence ofunprotected functional groups depends in part on the nature of theligation/condensation chemistry chosen so as to avoid or minimizepossible cross-reactions). The ligation methods can be used to produce alinker comprising a cleavage site for an enzyme. Moreover, the ligationmethods described herein can be used for the production of support boundcombination oligomers as well as arrays comprising one or morecombination oligomers.

All oligomer blocks of the combination oligomer can be peptide nucleicacid. Either, or both, of the terminal oligomer blocks or condensationoligomer blocks can contain at least one region of constant nucleobasesequence and at least one region containing variable nucleobasesequence. The constant nucleobase sequence can be between 1 to 10nucleobase containing subunits in length and the variable region can bebetween 3 to 8 nucleobase containing subunits in length.

A Method for Determining Binding of a Combination Oligomer to a BindingPartner

In certain other embodiments of this invention, a combination oligomeris formed that possesses a cleavage site for an enzyme wherein thecleavage site is protected from cleavage upon the binding of thecombination oligomer to a binding pair. Hence, this invention alsopertains to a method for determining whether or not a combinationoligomer binds to a possible binding partner (e.g. an aptmer or targetsequence). The method comprises contacting the combination oligomer andthe possible binding partner under suitable binding conditions tothereby possibly form a combination oligomer/binding partner complex.According to the method the combination oligomer is a polymer comprisinga segment of the formula: A-W-C, wherein A and C are oligomer blocksthat are optionally linked to other moieties and that are eachindependently a peptide nucleic acid, PNA chimera or PNA combinationoligomer. The group W is a linker of at least three atoms in length thatcovalently links oligomer block A to oligomer block C and that is acleavage site for an enzyme.

According to the method, the binding partner and the combinationoligomer are treated with an enzyme suitable for cleaving the cleavagesite under suitable enzyme cleaving conditions. Then a determination ismade of whether or not the combination oligomer has been cleaved by theactivity of the enzyme to thereby determine whether or not thecombination oligomer/binding partner complex formed.

Suitable enzyme cleavage conditions are those conditions under which theenzyme operates to act on a substrate. Numerous enzymes are commerciallyavailable and generally the product literature of the commercial vendorwill provide information on suitable enzyme cleavage conditions. Usingavailable information as well as routine experimentation it will bepossible for the ordinary practitioner to determine suitable enzymecleavage conditions.

According to the method, the enzyme will not substantially cleave thecombination oligomer provided that it binds to the binding partner.Thus, binding protects the combination oligomer from substantialdegradation by the enzyme. Consequently, the determination of binding ismade by analyzing for cleavage products such that if substantialcleavage products are detected, the combination oligomer must not havebound to the potential binding partner.

When the method involves the binding of a combination oligomer to atarget sequence, the hybridization will occur under suitablehybridization conditions wherein the combination oligomer hybridizesjuxtaposed on the target sequence such that there is no gap. The targetsequence can be in higher concentration than the combination oligomer sothat essentially all of the available combination oligomer is sequencespecifically bound if the target sequence of contiguous nucleobases ispresent.

Where the binding partner is a target sequence, and the combinationoligomer is bound to the target sequence, it is protected from theactivity of the enzyme. Accordingly, if the assay determines that thecombination oligomer is not substantially degraded, it must have boundto the target sequence (See: Example 4). Conversely, where thecombination oligomer was not protected from degradation, it can beconcluded that the target sequence was not present. It is also to beunderstood that since such an assay relies upon an enzymatic event,quantitation of the target sequence can be determined by determiningenzyme activity (e.g. measuring enzyme kinetics).

The oligomer blocks of the combination oligomer can all be peptidenucleic acid. The combination oligomer can be labeled or unlabeled,including without limitation, be a self-indicating combination oligomeror otherwise comprise on or more protected or unprotected functionalgroups. Moreover, the ligation methods described herein can be used forthe production of support bound combination oligomers as well as arrayscomprising one or more combination oligomers.

Kits of the Invention:

In still another embodiment, this invention pertains to kits. In oneembodiment said kit comprises two or more independently detectablecombination oligomers wherein each of said independently detectablecombination oligomers comprises a first oligomer block and a secondoligomer block that are each independently a peptide nucleic acid, a PNAchimera or PNA combination oligomer. The oligomer blocks are linkedcovalently to each other by a linker that is at least three atoms inlength. In each independently detectable combination oligomer, the firstand second oligomer blocks taken together encode a probing nucleobasesequence that is designed to sequence specifically hybridize to a targetsequence of contiguous nucleobases that is suitable for the formation ofa double stranded target sequence/combination oligomer complex. Theprobing nucleobase sequence in each independently detectable combinationoligomer differs from the probing nucleobase sequences of the otherindependently detectable combination oligomer(s) by at least onenucleobase. Each independently detectable combination oligomer containsat least one independently detectable label. The kit optionallycomprises; (i) one or more oligonucleotides; (ii) one or more buffers;(iii) one or more nucleotide triphosphates; (iv) a nucleic acidamplification master mix; or (v) one or more polymerase enzymes. In oneembodiment, the kit comprises at least two oligonucleotide primers.

According to the invention, the oligomer blocks of the combinationoligomers can all be peptide nucleic acid. The combination oligomers canbe labeled or unlabeled, including without limitation, be aself-indicating combination oligomers or otherwise comprise one or moreprotected or unprotected functional groups. In one embodiment, all ofthe combination oligomers of the kit are independently detectable,including being independently detectable self-indicating combinationoligomers. Optionally, the combination oligomers can comprise a linkercomprising a cleavage site for an enzyme. Moreover, the combinationoligomers can be support bound or be used in combination with the one ormore components of the kit to produce support bound combinationoligomers.

As indicated, the kits may optionally comprise one or more additionaloligomers, reagents or enzymes. In one embodiment, the one or moreadditional oligomers are primers suitable for performing a nucleic acidamplification reaction. Exemplary nucleic acid amplification processeshave been previously described herein. The kit can also comprisebuffers, one or more of nucleotide triphosphates, a polymerase enzymeand/or a nucleic acid amplification master mix, such as those typicallyused for PCR. In one embodiment, the kit can comprise two or moreindependently detectable self-indicating combination oligomers andreagents for determining one or more SNPs such as is described inExample 5, below.

Combination Oligomer Sets of the Invention:

In yet another embodiment, this invention pertains to a set of two ormore independently detectable combination oligomers. The combinationoligomers of the set each comprise a first oligomer block and a secondoligomer block that are each independently a peptide nucleic acid, PNAchimera or PNA combination oligomer. The oligomer blocks are linkedcovalently to each other by a linker that is at least three atoms inlength. In each independently detectable combination oligomer, the firstand second oligomer blocks taken together encode a probing nucleobasesequence that is designed to sequence specifically hybridize to a targetsequence of contiguous nucleobases to thereby form a double strandedtarget sequence/combination oligomer complex. The probing nucleobasesequence in each independently detectable combination oligomer differsfrom the probing nucleobase sequences of the other independentlydetectable combination oligomer(s) of the set by at least onenucleobase. Each independently detectable combination oligomer containsat least one independently detectable label. The set can comprise two ormore independently detectable self-indicating combination oligomerswherein each independently detectable combination oligomer comprises atleast one energy transfer set of labels such that at least one acceptormoiety of the energy transfer set is linked to one of the linkedoligomer blocks of the combination oligomer whilst at least one donormoiety of the energy transfer set is linked to another of the linkedoligomer blocks of the combination wherein the labels of the set arelinked to the oligomer blocks at positions that facilitate a change indetectable signal of at least one label when the combination oligomer ishybridized to a target sequence as compared to when the combinationoligomer is in a non-hybridized state.

According to the invention, the oligomer blocks of the combinationoligomers of a set can all be peptide nucleic acid. The combinationoligomers of a set can further comprise one or more protected orunprotected functional groups or one or more additional reportermoieties. Additionally, the combination oligomer of a set can comprise alinker comprising a cleavage site for an enzyme. Moreover, thecombination oligomers of a set can be support bound or be used incombination with one or more of the components of the kit to producesupport bound combination oligomers or arrays of combination oligomers.

Compound Libraries & Methods for Their Preparation:

In still another embodiment, this invention pertains to compoundlibraries. Although it is envisioned that in one respect the inventionpertains to one or more compound libraries comprising at least two setsof oligomer blocks (e.g. at least one set of terminal oligomer blocksand at least one set of condensation oligomer blocks), it is alsoenvisioned that terminal oligomer blocks and condensation oligomerblocks can be generated from the same compound library provided thateach member of a single set of oligomer blocks (bifunctional single setlibrary of oligomer blocks) is appropriately protected so that dependingupon the way the oligomer is pretreated prior to ligation, it can beused to produce either of the required terminal block or condensationblock. Whilst the latter approach can be advantageous because it limitsthe absolute number of oligomers required for the library, it also addsone or more additional steps to the preparation of the combinationoligomers. Accordingly, this invention pertains to both one or morebifunctional single set libraries as well as one or more librariescomprising two or more oligomer block sets.

This invention is also directed to a method for forming a terminaloligomer block and a condensing oligomer block from a bifunctionalsingle set library. The method comprises providing a bifunctional singleset library of at least two oligomer blocks. One oligomer block of thebifunctional single set library is treated to thereby remove one or moreof the protecting groups to thereby produce a terminal oligomer. Oneoligomer block of the bifunctional single set library is also treated toremove one or more different protecting groups, as compared with thosethat produce the terminal oligomer block, to thereby produce acondensing oligomer block.

According to the invention, the various functional groups of theoligomer blocks of the bifunctional single set library can be orthogonalsuch that they can be selectively removed without deprotecting othergroups. Examples of such orthogonal protecting schemes are well known inpeptide chemistry, nucleic acid chemistry and PNA chemistry. Forexample, PNA monomers typically use the acid labilebenzhydroloxycarbonyl (Bhoc) to protect the nucleobases of the PNAmonomers, whereas the base labile fluorenylmethoxycarbonyl (Fmoc) groupis used to protect the amine of the PNA monomers. It is to be understoodthat the ordinary practitioner will be able to prepare orthogonallyprotected oligomer blocks suitable for use in the previously describedmethod using no more that routine experimentation.

Thus, this invention is also directed to a compound library comprising abifunctional single set of oligomer blocks. The oligomer blocks of theset can be used to produce both terminal oligomer blocks andcondensation oligomer blocks by the removal of certain protectinggroups. The oligomer blocks of the bifunctional set are peptide nucleicacid oligomer, PNA chimera or PNA combination oligomer. The oligomerblocks of the bifunctional set are selected to comprise functionalmoieties that form a linker of at least three atoms in length when aterminal oligomer block is condensed with a condensation oligomer block.Furthermore, the oligomer blocks are not support bound and do notcomprise nucleobase-protecting groups.

In still another embodiment, this invention pertains to another compoundlibrary. According the invention, this compound library comprises atleast one set of terminal oligomer blocks and at least one set ofcondensing oligomer blocks wherein each set of blocks comprises two ormore different oligomers and said oligomer blocks are selected from thegroup consisting of: peptide nucleic acid oligomer, PNA chimera and PNAcombination oligomer. The oligomer blocks are selected to comprisefunctional moieties that form a linker of at least three atoms in lengthwhen a terminal oligomer block is condensed with a condensation oligomerblock. Additionally, the oligomer blocks are not support bound and theoligomer blocks do not comprise nucleobase-protecting groups. It is tobe understood that a compound library of this invention is not to belimited to one or two sets of block oligomers. By way of a non-limitingexample, the library may comprise three or more sets of oligomer blocks.

Accordingly, in still another embodiment, this invention pertains toanother compound library. The compound library comprises at least oneset of terminal oligomer blocks and at least two sets of condensingoligomer blocks. According to the invention, each set of oligomer blockscomprises two or more different oligomers and the oligomer blocks ofeach set are independently peptide nucleic acid oligomer, PNA chimera orPNA combination oligomer. The oligomer blocks are selected to comprisefunctional moieties that form a linker of at least three atoms in lengththat covalently links the oligomer blocks when a terminal oligomer blockis condensed with a condensation oligomer block. Additionally, theoligomer blocks are not support bound and the oligomer blocks do notcomprise nucleobase-protecting groups. Furthermore, all of the oligomerblocks of a set of condensing oligomer blocks contain the sameindependently detectable reporter moiety wherein different sets ofcondensation oligomer blocks comprise different independently detectablelabels. All of the oligomer blocks of the at least one set of terminaloligomer blocks comprise the same non-fluorescent quencher moiety.

A compound library of this configuration is particularly useful for theproduction of pairs of independently detectable self-indicatingcombination oligomers. For example, one terminal oligomer block fromeach set of independently detectable oligomer blocks can be ligated tothe same or different terminal oligomer block comprising thenon-fluorescent quencher moiety. Such pairs of independently detectableself-indicating combination oligomers can be used to in SBP analysis.Moreover, the library can be used for generating pairs of combinationoligomers for SNP analysis such as is described in Example 5.

It is to be understood that the length of the oligomer blocks of alibrary is not a limitation. For example, the oligomer blocks of alibrary can be independently selected to be trimers, tetramers,pentamers, hexamers, heptamers or octamers. Moreover, the oligomerblocks of a set need not be all of the same length, label configurationor the like. The oligomer blocks of a set can comprise both variable andconstant regions of nucleobases. It is also to be understood that theoligomer blocks of a library can be support bound. Optionally, thelibrary may itself exist as an array of block oligomers that can be usedto form combination oligomers by the process of ligating/condensing oneor more oligomer blocks thereto.

According to one embodiment, a terminal oligomer block of a library canbe condensed with a condensation oligomer block of the same or anotherlibrary to thereby form a combination oligomer of desired nucleobasesequence. Accordingly, ligation/condensation of the oligomer blocks ofthis invention produce a linker that is at least three atoms in lengthand that links two oligomer blocks. The linker may comprise a cleavagesite for an enzyme. Possible linkers have been previously describedherein. Non-limiting methods for forming combination oligomers by theligation/condensation of oligomer blocks have previously been describedherein. Such methods can be used in combination with the librariesdescribed herein. It is an advantage that nucleobases of the oligomerblocks do not need to be protected to be useful in saidligation/condensation processes.

In accordance with this invention, the nucleobase sequence of theterminal oligomer block and nucleobase sequence of the condensingoligomer block can be chosen from the sets of a library, or libraries,of oligomer blocks to thereby enable the rapid, efficient and/orappropriately scaled synthesis of a combination oligomer that issuitable for a chosen application. Thus, the possession of a library, orlibraries, of oligomer blocks can facilitate the rapid, efficient and/orappropriately scaled synthesis of numerous combination oligomers ofdifferent but known nucleobase sequence, wherein the number of potentialcombination oligomers of different nucleobase sequence that can possiblybe made from a library is determined by the diversity of the terminaloligomer block set and condensing oligomer block set (or the diversityof a single oligomer block set as appropriate) and wherein the diversityof a set will depend on the number of oligomers of different nucleobasesequence in the set.

The diversity of a set of oligomer blocks is determined by the number ofpossible variables at a position raised to the power of the number ofvariable sites. For example a set of pentamer blocks, wherein each ofthe five subunits could be variable and the possible variations includedthe four natural nucleobases A, C, G & T, would produce an all inclusiveset of 4⁵=1024 pentamers. However, some or all of the oligomer blocks ofone or both of the terminal oligomer blocks of a set and the condensingoligomer blocks of a set may comprise both a region of constantnucleobase sequence as well as a region of variable nucleobase sequence.Thus, a complete set of oligomer blocks consisting of only the fournatural nucleobases A, C, G & T is 4^(n) wherein n is a whole numberrepresenting only the number of variable nucleobase positions. Howeverthere is no requirement that a library of this invention comprise acomplete set of block oligomers or that the sets of block oligomers of alibrary, or libraries, all be of the same length or diversity. Thus, incertain embodiments, a library can comprise: 1) oligomer blocks whereinless than all four of the natural nucleobases are present; 2) oligomerblocks wherein all four of the natural nucleobases are present; 3)either of 1) or 2) further comprising one or more non-naturallyoccurring nucleobases; or 4) oligomer blocks only comprisingnon-naturally occurring nucleobases.

In fact use of the following non-natural nucleobases is specificallycontemplated: 5-propynyl-uracil, 2-thio-5-propynyl-uracil,5-methylcytosine, pseudoisocytosine, 2-thiouracil and 2-thiothymine,2-aminopurine, N9-(2-amino-6-chloropurine), N9-(2,6-diaminopurine),hypoxanthine, N9-(7-deaza-guanine), N9-(7-deaza-8-aza-guanine) andN8-(7-deaza-8-aza-adenine). Binding pair motifs for these nucleobaseshave also been previously described herein.

In one embodiment, the number of variable positions (variable nucleobasecontaining subunits) in a set of block oligomers is 3-8 such that thenumber of oligomers in a complete set (A, C, G & T as the nucleobases)is 64, 256, 1024, 4096, 16384 and 65536, respectively.

In one embodiment, a region of constant nucleobase sequence can bepresent when the oligomers of the set have a common utility whereinvariation is required in only a subsection in order to perform a desiredexperiment or examination. For example, the constant region can be inthe range of 1-10 nucleobase containing subunits.

In one embodiment, the terminal oligomer blocks of a set are peptidenucleic acid oligomers having a C-terminal amide and an N-terminalnatural amino acid. For example, the N-terminal amino acid can beselected from the group consisting of: lysine, cystine, glutamic acidand aspartic acid. In another embodiment, the N-terminal natural aminoacid is glycine.

In another embodiment, the terminal oligomer blocks of the set arepeptide nucleic acids having a C-terminal amide and an N-terminalreactive moiety such as aminooxyacetyl, 2-thioacetyl 3-thiopropionyl,malimido and haloacetyl and vinyl. See FIGS. 29-31 for an illustrationof the operation of these functional groups in view of theligation/condensation chemistry previously discussed.

In still another embodiment, the set of condensation oligomer blocks arepeptide nucleic acids having a C-terminal natural amino acid moiety anda capped or protected N-termini (e.g. labeled). The C-terminal aminoacid can be selected from the group consisting of: lysine, cystine,glutamic acid and aspartic acid. In another embodiment, the C-terminalamino acid is glycine.

In still another embodiment, the terminal oligomer blocks of the set arepeptide nucleic acids having a capped or protected N-termini and aC-terminal reactive moiety such as N-hydroxysuccinimidyl, haloacetyl oraldehyde moiety or a functional group selected from the group consistingof: chloroacetyl bromoacetyl, iodoacetyl, glycinal and β-alinal. SeeFIGS. 29-31 for an illustration of the operation of these functionalgroups in view of the condensation/ligation chemistry discussedpreviously.

Non-Limiting Examples of the Sets of a Library

A exemplary library can comprise three or more sets of oligomer blockswherein at least two sets can be substantially identical except for thenature of the label such that the two or more different labeling schemes(one labeling scheme being common to each individual set of oligomerblocks) renders each set of oligomer blocks independently detectable.The two or more independently detectable oligomer block sets may be setsof terminal oligomer blocks or condensation oligomer blocks. Byproducing, two sets of oligomer blocks that are essentially identicalbut for the nature of the attached independently detectable label, it ispossible to prepare pairs of independently detectable combinationoligomers.

According to the invention, at least two oligomer blocks comprisingindependently detectable labels can be ligated to the same or differentoligomer blocks that, for example, are labeled with an acceptor orquencher moiety. For example the label can be a non-fluorescent quenchermoiety that is suitable for substantially quenching the detectability ofeach of the different independently detectable labels such that whenoligomer blocks are ligated to form a combination oligomer, pairs ofindependently detectable self-indicating probes can be formed based uponthe nature of the oligomer blocks that are chosen from the libraries forligation. Example 6 of this specification illustrates the ligation oflabeled oligomer blocks to thereby produce pairs of independentlydetectable combination oligomer probes suitable for use in SNPgenotyping.

If the acceptor or quencher moiety is not present on the oligomer blockto which the independently detectable blocks are ligated, then thecombination oligomers formed by the ligation will be a labeledcombination oligomer wherein the choice of labels will be limited onlyby the diversity of the previously described two or more sets ofindependently detectably labeled oligomer blocks. It is also noted thatthe library may optionally comprise both the unlabeled set of oligomerblocks as well as the set of oligomer blocks comprising a linkedacceptor or quencher moiety so that depending on the requirements,either a fluorescently labeled independently detectable combinationoligomer or a self-indicating combination oligomer can be prepared fromthe ligation reaction.

In another preferred embodiment, the set described above can furthercomprise an additional set of unlabeled oligomers that is substantiallyidentical to the sets comprising the independently detectable labelsexcept that they are unlabeled. Hence, when combined, through ligation,with an oligomer from the other set of unlabeled oligomer blocks, acompletely unlabeled combination oligomer can be prepared. Suchunlabeled oligomers can, for example, be used as blocking probes (Seefor Example: U.S. Pat. No. 6,110,676), used as capture probes, used asdetector probes wherein a labeled antibody is used to detect the hybrid(See for Example: U.S. Pat. No. 5,612,458); or used to produce an arrayof unlabeled combination oligomers.

TABLE 1 Configuration Of Potential Oligomer Block Sets Of A LibraryProperties of Combination Condensation Oligomer/Potential Block SetTerminal Block Set Applications Unlabeled Unlabeled Unlabeled probe orprimer; blocking probe, capture probe or detector probe used incombination with antibody detection methods Unlabeled Label Labeledprobe or primer Label Unlabeled Labeled probe or primer Donor/AcceptorDonor/Acceptor Self-Indicating Probe Donor/Acceptor Unlabeled ComponentPolymer of Detection Complex Unlabeled Donor/Acceptor Component Polymerof Detection Complex

In accordance with the prior description, Table 1 summarizes variouspossibilities for the make up of sets of oligomer blocks of a possiblelibrary as well as the properties of the combination oligomers preparedby the ligation thereof. Of course Table 1 is not intended to beexhaustive of possibilities and, as discussed herein, oligomer blocksets comprising essential identical oligomers, except for the labelattached thereto, can be an important aspect of this invention and willexpand the utility of the library. One or more of the sets of oligomerblock can also optionally contain protected or unprotected functionalgroups linked to the oligomer blocks at the termini or linked at aposition internal to the oligomer blocks (or when ligated, at a positionintegral to the linker of the combination oligomer). In this regard, theoligomer blocks can be labeled either pre- or post-ligation, dependingon a practitioner's desire and available resources or otherwise thefunctional groups can be used to attach the oligomer blocks or formedcombination oligomers to a surface.

Arrays of Combination Oligomer & Methods for Their Preparation:

Arrays comprising nucleic acid, peptide nucleic acid or chimera havebeen described in the literature. Generally, a nucleic acid or peptidenucleic acid can be immobilized to an array by either synthesizing theoligomer on the support or otherwise by immobilizing the previouslyformed oligomer. For example, the condensation oligomer blocks can beligated to one or more of support bound terminal oligomer blocks tothereby form an array of combination oligomers suitable for performing adesired application. Alternatively, a preformed combination oligomer canbe reacted at a position of an array to thereby effect the attachment ofthe combination oligomer.

Because the location and sequence of each support bound oligomer isknown, arrays can be used to simultaneously detect, identify and/orquantitate the presence or amount of one or more target sequences in thesample. For example, a target sequence can be captured by thecomplementary combination oligomer on the array surface and then theprobe/target sequence complex is detected. Since the composition of thecombination oligomer is known at the location on the surface of thearray (because the oligomer was synthesized or attached to this positionof the array), the composition of target sequence(s) can be directlydetected, identified and/or quantitated by determining the location ofdetectable signal generated on the array. Thus, arrays may be useful indiagnostic applications or in screening compounds for leads that mightexhibit therapeutic utility.

In yet another embodiment, this invention pertains to an array of atleast two combination oligomers wherein at least one of the combinationoligomers comprises a segment having the formula: A-B-C. According tothe invention, oligomer blocks A and C are each independently a peptidenucleic acid, PNA chimera or PNA combination oligomer and are optionallylinked to other moieties. The linker B is at least three atoms in lengthand covalently links oligomer block A to oligomer block C. Oligomerblocks A and C can be unlabeled, labeled with one or more reportermoieties or comprise one or more protected or unprotected functionalgroups linked thereto. Oligomer blocks A and C taken together encode aprobing nucleobase sequence that is designed to sequence specificallyhybridize to a target sequence of contiguous nucleobases to thereby forma double stranded target sequence/combination oligomer complex.

In yet another embodiment, this invention pertains to methods forforming an array of combination oligomers. In one embodiment, the methodcomprises reacting, at a site on a solid carrier, a first oligomerblock, a second oligomer block, and optionally a condensation reagent orreagents under condensation conditions to thereby form a combinationoligomer having a linker of at least three atoms in length thatcovalently links the first oligomer block to the second oligomer block.According to the invention, one of said two oligomer blocks is supportbound. Further, the first and second oligomer blocks are eachindependently a peptide nucleic acid oligomer, PNA chimera or PNAcombination oligomer. Additionally, one or both oligomer blocks do notcomprise nucleobase protecting groups and the combination oligomer formsin the absence of a template. The method further comprises repeating themethod with one or more different oligomer blocks at one or moredifferent sites until the desired array of combination oligomers isconstructed.

In still another embodiment, this invention pertains to another methodfor forming an array of combination oligomers. The method comprisesreacting, at a site on a solid carrier, a functional group of acombination oligomer having a linker of at least three atoms in lengththat covalently links the first oligomer block to the second oligomerblock with a surface functional group to thereby covalently attach thecombination oligomer to the surface. According to the method, the firstand second oligomer blocks of the combination oligomer are eachindependently a peptide nucleic acid oligomer, PNA chimera or PNAcombination oligomer. Moreover, one or both oligomer blocks do notcomprise nucleobase-protecting groups. The method further comprisesrepeating the method for attachment of the combination oligomer with oneor more different combination oligomers at one or more different sitesuntil the desired array of combination oligomers is constructed.

EXAMPLES

This invention is now illustrated by the following examples that are notintended to be limiting in any way.

General Information on PNA and DNA Synthesis/Oligomers

All PNA Oligomers were prepared from commercial reagents andinstrumentation obtained from Applied Biosystems, Foster City, Calif.using manufacturer published procedures, other well-known procedures orthose disclosed in published PCT applications WO99/21881, WO99/22018 andWO99/49293.

All nucleic acid (DNA) oligomers, used as target sequence, were preparedfrom commercial reagents and instrumentation obtained from AppliedBiosystems, Foster City, Calif. using manufacturer published proceduresor were obtained from commercial vendors of custom oligonucleotides.

Example 1 Evaluation Of Combination Oligomers

Introduction & Purpose

A series of PNA oligomers that contained the same nucleobase sequence(coding for a variable region in the human K-ras gene) but that wereeither native (unmodified) or else comprising two oligomer blockscentrally linked by various linker moieties (a “combination oligomer”)were designed and synthesized for the purpose of evaluatinghybridization properties (See: Table 2). The oligomers were synthesizedusing standard methodologies, without any ligation steps. Criticalfactors that were evaluated included: 1) whether or not a gap should bepresent between the oligomer blocks when hybridized to a targetsequence; 2) whether or not labels that were attached to the combinationoligomers would either: (i) influence hybridization properties; or (ii)effectively function in the manner for which they are typically designed(e.g. would the energy transfer set allow for determination of oligomerhybridization by exhibiting a change in detectable signal as comparedwith unhybridized oligomer); 3) how do the hybridization properties ofthe combination oligomers otherwise compare with the native oligomers;and 4) whether or not there is a preferred embodiment for a combinationoligomer. In order to evaluate the hybridization properties of thenative and combination oligomers, a series of fully of partiallycomplementary nucleic acid (DNA) target sequences were likewise obtained(See: Table 3).

Description of Tables 2 & 3

With reference to Table 2, all of the native and combination oligomersthat were evaluated are identified as PNA A through PNA L (column I).The complete sequence of each probe is illustrated in column II. Incolumn III, the particular linker type is identified and in column IVthe presence or absence of reporter moieties is identified.

Again with reference to Table 2, the native oligomers are PNA F and PNAL. By native we mean that the nucleobase sequence is continuous withoutinclusion of a linker that links two backbone subunits of two componentoligomer blocks. For this Example, the backbone that is native to thepolymer is continuous without interruption. PNA F differs from PNA L inthat PNA L is labeled with a fluorophore and quencher moiety asdescribed in WO99/21881 (i.e. it is a self-indicating oligomer probe)but PNA F is unlabeled. It is noted that the oligomers PNA A to PNA Elikewise differ from PNA G to PNA K, in this respect. Consequently, PNAA through PNA F differ from PNA G to PNA L only in the absence orpresence of reporter moieties, respectively. PNA A to PNA E arecombination oligomers comprising different linkers. PNA G to PNA Kcomprise the same linkers as PNA A to PNA E, respectively.

TABLE 2 Table Of Native & Combination Oligomers Prepared I II III IVProbe ID Sequence Linker Label PNA A H-ACCAG-X-TCCAA-NH₂ X-linker NoLabel PNA B H-ACCAG-E-TCCAA-NH₂ E-linker No Label PNA CH-ACCAG-GlyGly-TCCAA-NH₂ 2 Glycines No Label PNA D H-ACCAG-O-TCCAA-NH₂ 1O-linker No Label PNA E H-ACCAG-OO-TCCAA-NH₂ 2 O-linkers No Label PNA FH-ACCAGTCCAA-NH₂ No Linker No Label PNA G F-Glu-ACCAG-X-TCCAA-K-K-D-NH₂X-linker SI PNA H F-Glu-ACCAG-E-TCCAA-K-K-D-NH₂ E-linker SI PNA IF-Glu-ACCAG-GlyGly-TCCAA-K-K-D-NH₂ 2 Glycines SI PNA JF-Glu-ACCAG-O-TCCAA-K-K-D-NH₂ 1 O-linker SI PNA KF-Glu-ACCAG-OO-TCCAA-K-K-D-NH₂ 2 O-linkers SI PNA LF-Glu-ACCAGTCCAA-K-K-D-NH₂ No Linker SI For Table 2, all PNA sequencesare written from the amine to the carboxyl terminus (left to right) inaccordance with convention used for the illustration of peptidesequences. Other abbreviations are: F = 5-(6)-carboxyfluorescein, D =4-((4-(dimethylamino)phenyl)azo)benzoic acid (dabcyl), O or O-linker =8-amino-3,6-dioxaoctanoic acid; K = the amino acid L-Lysine, Glu = theamino acid L-glutamic acid, Gly = the amino acid glycine, E or E-linker= the moiety referred to as U.S. Pat. No. 6,326,479 and X or X-linker =the moiety referred to as “E” in U.S. Pat. No. 6,326,479; SI =self-indicating.

With reference to Table 3,the nucleobase sequence of several nucleicacid target sequences (possible binding partners) that potentially bindto the native and combination oligomers of Table 2 are illustrated. Thedifferent nucleic acid oligomers are identified in column I. Thenucleotide sequence of the different nucleic acid oligomers isidentified in column II. A description of the nature of the nucleobasesequence is provided in column III. Column IV merely denotes theassigned Seq. Id. No.

Nucleic acid oligomers DNA P and DNA Q differ by inclusion of theitalicized and underlined G residue (DNA P). This residue represents the“gap base” or “gap” that is designed to be present when the native andcombination oligomers are allowed to hybridize to the target sequences.However, because this gap base is not present when the native andcombination oligomers hybridize to nucleic acid oligomers DNA Q-V, thereis no “gap” (See FIG. 1). Nucleic acid oligomer DNA Q represents theperfect complement to the native and combination oligomers of Table 1.Nucleic acid oligomers DNA R-T comprise a mismatch (highlighted in boldtext) as compared with the perfectly complementary oligomer DNA Q,wherein the mismatch is centrally located with respect to one of the5-mer oligomer blocks. Nucleic acid oligomers DNA U and DNA V comprise amismatch (highlighted in bold text) as compared with the perfectlycomplementary oligomer DNA Q, wherein the mismatch is not centrallylocated with respect to one of the 5-mer oligomer blocks.

TABLE 3 Table Of Target Sequences I IV Oligo II III Seq. ID SequenceDescription ID No. DNA P TAGTTGGA G CTGGTGGC Match with gap 1 DNA QTAGTTGGACTGGTGGC Match contiguous 2 DNA R TAGTTGGACTTGTGGC MisMatchcontiguous 3 DNA S TAGTTGGACTAGTGGC MisMatch contiguous 4 DNA TTAGTTGGACTCGTGGC MisMatch contiguous 5 DNA U TAGTTGGACGGGTGGC MisMatchcontiguous 6 DNA V TAGTTGGAGTGGTGGC MisMatch contiguous 7Tm Experiments with Combination Oligomers Hybridized to the Nucleic AcidTarget Sequences

In a thermal melting experiment, a hybrid between two polymer strandsis, by heating, melted into its component oligomers. The temperature atwhich the dissociation of the hybrid occurs is a function ofthermodynamic properties that are characteristic of that particularhybrid. The temperature at which half of the hybrids remain doublestranded, with the remaining half existing as single strands, iscommonly referred to as the Tm of the hybrid.

In order to perform the Tm experiments, samples were prepared by mixing0.5 μM of each of the native or combination oligomers (Table 2) withcertain of the nucleic acid target sequences (Table 3) in 2.5 mL TmBuffer (100 mM sodium chloride, 10 mM potassium phosphate, pH 7.1) inquartz cuvettes. The native and combination oligomers were also testedin the absence of target sequences (“No DNA” control) as a means todetermine whether or not the combination oligomers were self-annealingor self-complementary. In no case was there a Tm in the absence of atarget sequence, thereby indicating that the native and combinationpolymers do not self-anneal or self-hybridize. The Tm analyses wereperformed using the Cary 100 spectrometer, using the “Thermal” programsupplied by the instrument manufacturer (Varian). All absorbancemeasurements were recorded with reference to a blank containing buffer.All analyses were performed in dual beam mode.

Prior to each measurement, samples were denatured by heating to 95° C.for 2 minutes, then cooled to 25° C. For all Tm determinations, thesamples were heated and then cooled at a rate of 0.5 degrees per minute.The beginning and end points for each melt were determined empiricallyprior to each run, depending upon the Tm observed during the pre-melt.Throughout the experiments, absorbance data were recorded at 0.5 degreeincrements. The Tm values were then calculated using instrument softwareand a moving average taken over 10.5 degrees (corresponding to 21 datapoints at 1 point per 0.5 degree). The Tm data for various experimentsare displayed in Tables 4 and 5. All Tm values presented are the averageof the values obtained from the heating and cooling steps.

Discussion of Data Table 4; Determining Optimal Probe Design

TABLE 4 Table Of Tms For Determining Optimal Probe Design I II III IVProbe ID DNA P DNA Q ΔTm P − Q PNA A 29.0 37.4 8.3 PNA B 30.9 37.3 6.4PNA C 25.2 41.5 16.3 PNA D 23.3 40.3 17.0 PNA E 21.2 36.1 14.9 PNA F35.2 53.1 17.9 PNA G 30.4 37.1 6.7 PNA H 30.5 37.1 6.6 PNA I 23.6 41.818.2 PNA J 23.1 40.2 17.1 PNA K 19.6 36.1 16.5 PNA L 38.8 53.2 14.4

Table 4 summarizes Tm data for the hybridization of all of native andcombination oligomers PNA A to PNA L with either of the gap or no gapnucleic acid target sequences DNA P and DNA Q, respectively (See: FIG.1). With reference to Table 4, the native or combination oligomer usedto determine the Tm is identified in column I. In column II, the Tm (°C.) for all hybrids formed with nucleic acid target sequence DNA P isrecorded. In column III, the Tm for all hybrids formed with nucleic acidtarget sequence DNA Q is recorded. In column IV, the difference in Tm(ΔTm P-Q in ° C.) is recorded for hybrids using DNA P vs. DNA Q,respectively.

Again with reference to Table 4, for every hybrid the Tm value isgreater if DNA Q is used as compared with DNA P. The absolute differencein Tm for a native or combination oligomer is found in column IV.Because the difference between DNA P and DNA Q is the presence orabsence of a gap, respectively, it is clear that the more stable hybridis always formed when the block oligomers hybridize juxtaposed to thetarget sequence such that there is no gap. Moreover, the substantialdifference in Tm (6.4 to 17.9° C.) indicates that this is asubstantially preferred hybrid as compared with hybrids comprising agap.

Again with reference to Table 4, it is clear that the data for PNA A toPNA F is substantially the same as for PNA G to PNA L, respectively. Thestrong similarity in data for each unlabeled PNA as compared with theequivalent labeled PNA indicates that the labels do not substantiallyaffect the hybridization properties of the various native andcombination oligomers.

Discussion of Data Table 5; Determining Probe Discrimination/Selectivity

Because PNA C appeared to have the most preferred linker, we selectedthe combination oligomer PNA C for further comparison with the nativeoligomer PNA F. Table 5 summarizes Tm data for the hybridization of eachof PNA C and PNA F with each of the nucleic acid target sequences DNA Qto DNAV; wherein in all cases the hybrid comprises no gap. Withreference to Table 5, the nucleic acid target sequence used to determinethe Tm is identified in column I. In column II, the Tm (° C.) for allhybrids formed with PNA C is recorded. In column III, the Tm for allhybrids formed with PNA F is recorded. In column IV, the difference inTm (ΔTm in ° C.) for the hybridization between the perfect complement(DNA Q to PNA C) and the Tm recorded for a particular nucleic acidtarget comprising a mismatch (point mutation of single nucleotidepolymorphism) and PNA C is recorded. In column V, the difference in Tm(ΔTm in ° C.) for the hybridization between the perfect complement (DNAQ to PNA F) and the Tm recorded for a particular nucleic acid targetcomprising a mismatch (point mutation of single nucleotide polymorphism)and PNA F is recorded. In column VI, the absolute difference in thevalues in column IV as compared to column V (ΔC−ΔF), is recorded.

TABLE 5 Table Of Tms For Determining Probe Discrimination/Selectivity IIIII IV V VI I PNA C PNA F ΔC ΔF ΔC − ΔF DNA Q 41.5 53.1 — — 0.0 DNA R21.0 34.0 20.5 19.1 1.3 DNA S 19.7 36.0 21.8 17.1 4.7 DNA T 19.5 35.022.0 18.1 3.9 DNA U 20.3 35.0 21.2 18.1 3.1 DNA V 21.8 32.0 19.7 21.1−1.4

Because the only perfectly complementary hybrid is formed using DNA Q,all data for each different PNA hybridizing to a mismatch containingnucleic acid target sequence is compared with DNA Q. This is why thereis no data in columns IV to VI for DNA Q.

With reference to column IV of Table 5, the ΔC value for DNA R to DNA Tis 20.5-22.0° C. This indicates that a mismatch centrally located in oneof the block oligomers of the combination oligomer imparts a substantialdestabilization effect to the hybrid. By comparison, the ΔF value forDNA R to DNA T is 17.1-19.1° C. (See column V). This indicates that amismatch in the same position of a hybrid formed from a native oligomerand a target sequence imparts a lesser destabilization effect ascompared with the most nearly equivalent hybrid formed using acombination oligomer (having a gly-gly dimer linker; See column VI) anda target sequence. Although there is clearly a small difference in ΔTmthat occurs as a result of the nature of the particular nucleobaseforming the mismatch, the data includes all possible natural mismatchcombinations. The data indicates that in all cases, the destabilizingeffect is greater for the combination oligomer as compared with thenative oligomer (See column VI).

With reference to column IV of Table 5, the ΔC value for DNA U is 21.20°C. By comparison, the ΔF value for DNA U is 18.1° C. (See column V).This indicates that a mismatch located non-centrally in one of the blockoligomers of the combination oligomer imparts a substantialdestabilization effect to the hybrid that is again more significant ascompared with the destabilizing effect resulting from the inclusion ofthe same mismatch in a hybrid using a native oligomer and the sametarget sequence (See column VI). This data suggests that the mismatchneed not be centrally located within the oligomer block of thecombination oligomer in order to achieve a benefit indiscrimination/selectivity as compared with a native oligomer.

With reference to column IV of Table 5, the ΔC value for DNA V is 19.7°C. By comparison, the ΔF value for DNA U is 21.1° C. (See column V).This indicates that a mismatch located at the termini of one of theblock oligomers of the combination oligomer imparts a lesserdestabilization effect to the hybrid as compared with the destabilizingeffect resulting from the inclusion of the same mismatch in a hybridusing a native oligomer and the same target sequence (See column VI).This data suggests that the mismatch located at the terminus of theoligomer block of the combination oligomer does not result in improveddiscrimination/selectivity as compared with a native oligomer.

Fluorescence Experiments with Self-Indicating Combination Oligomers

In order to evaluate whether or not the self-indicating combinationoligomers (PNA G through L), could operate in the manner expected (basedupon the label configuration), they were hybridized to the complementarynucleic acid, DNA Q, and the resulting fluorescence of each sample wasmeasured.

The self-indicating combination oligomers were diluted to a finalconcentration of 1.0 μM in 100 μL of Tm Buffer and placed intoindividual wells of a 96 well polyethylene microtitre plate. Thefluorescence of each self-indicating combination oligomer was thendetermined using a Wallac Victor fluorescent plate reader, using afilter set that is optimized for detecting the fluorescence offluorescein. After recording this baseline value for eachself-indicating combination oligomer, a 3 μL aliquot containing 35 μMDNA Q was added to each well thereby resulting in a final concentrationof 1.0 μM. The sample in each well was then mixed and hybridization wasallowed to proceed for 10 minutes. As a control a non-complementarytarget was added in a similar way to another complete set ofself-indicating combination oligomers. In every case, the fluorescentvalue for each self-indicating combination oligomer increasedsignificantly when the complementary DNA was added but did notappreciably increase in the presence of the non-complementary DNA.

Summary

In summary, the data supports the following conclusions: 1) it ispreferable that there be no gap between the oligomer blocks whenhybridized to a target sequence since this embodiment results in themost stable hybrid and there is an improved selectivity/discriminationobserved for such oligomers; 2) when labels are attached to thecombination oligomers they do not appear to either: (i) substantiallyinfluence hybridization properties; or (ii) function in the manner thatdiffers from that which they are typically designed; 3) although the Tmof combination oligomers tend to be lower than the native oligomers, thecombination oligomer can be designed to exhibit a significantly enhancedselectivity/discrimination as compared with the native oligomers; and 4)the most preferred embodiment for a combination oligomer appears to bean oligomer comprising a amino acid dimer, and in particular the gly-glydimer.

Example 2 Block Ligation in the Absence of a Template

Introduction & Purpose

The preceding example indicated that combination oligomers could beuseful in hybridization reactions used, for example, to detect a targetnucleic acid sequence. In this Example, the goal was to determinewhether or not it would be possible to efficiently ligate two oligomerblocks in the absence of a template (i.e. non-template directedligation) to thereby form a combination oligomer. The ligation reactionwas intended to produce a gly-gly dimer linker.

Reagent Solutions:

-   -   0.5M 2-(N-Morpholino)ethane sulfonic acid (MES) Buffer; pH 4.5    -   HOAt Solution: prepared by mixing 21.4 mg of        1-Hydroxy-7-azabenzotriazole (HOAt) with 2 mL of 1:1,        N,N′-dimethylformamide (DMF): 0.5M MES Buffer pH 4.5.    -   EDC Solution was prepared by adding 10 mg        1-Ethyl-3-(3-Dimethylamino-propyl)carbodiimide hydrochloride        (EDC) to 100 μL of 0.5M MES Buffer (this solution should be        prepared immediately before use).    -   Quenching Solution was prepared by adding 10 mg of        Glycinamide-HCl to 100 μL of 1M sodium bicarbonate solution        (unbuffered).        General Reaction Conditions:

Final reaction conditions are generally 10 equivalents of HOAt and 500equivalents of EDC in a reaction mixture that was 750 μM in eacholigomer block to be ligated. It is noted that in these experiments, notemplate was used.

Experiment One:

About 19.1 nmol of each PNA oligomer (the oligomer blocks to be ligated)was added to a single 0.5 mL microcentrifuge tube and subsequentlydried. To the dried PNA oligomer mixture was added 4.8 μL of 1:1DMF:water. This solution was mixed by vortex and then 2.43 μL of HOAtSolution and 18.3 μL of EDC Solution was added. After 5 min, 10 μL ofthe reaction mixture was removed and quenched with 10μL of the QuenchingSolution. The product was then analyzed by Maldi-TOF mass spectrometryand HPLC. After one hour, 10 μL of the remaining reaction mixture wasquenched in the same way and analyzed by Maldi-TOF mass spectrometry andHPLC. The remaining 5.5 μL of the reaction mixture was quenched with 30%aqueous ammonium hydroxide and analyzed by Maldi-TOF mass spectrometryand HPLC.

Experiment Two:

About 95.6 nmol of each PNA oligomer (the oligomer blocks to be ligated)was added to a single 0.5 mL microcentrifuge tube and subsequentlydried. To the dried PNA oligomer was added 23.7 μL of 1:1 DMF:water. Thesolution was mixed by vortex and then 12.2 μL of HOAt Solution was addedfollowed by the addition of 91.6 μL of EDC Solution. The reaction wasquenched after one hour with 127.5 μL of Quenching Solution. The productwas then analyzed by Maldi-TOF mass spectrometry and HPLC.

Results:

TABLE 6 Table Of Ligation Results Experiment # % Product (5 min.) %Product (1 hour) 1 71.9 82 2 No Data 85

For both experiments the condensing block was capped with an acetylmoiety. Specifically, the condensing block (PNA) was Ac-ACC-AG-Gly-COOHand the terminal block was H-Gly-TCC-AA-NH₂ wherein Ac represents theacetyl cap, Gly represents that amino acid glycine and otherabbreviations are well known in the field of peptide chemistry.

With reference to Table 6, the percent completion of the ligationreaction was measured based on the integration of peak area (excludingthe peak representing the HOAt) of the HPLC analysis of the product. Theresults demonstrated that the two PNA oligomer blocks can, in theabsence of a template, be successfully ligated in greater than 70% yieldwithin 5 minutes but also show that a longer reaction time will lead toa greater percentage of product formation. The reaction occurring asexperiment number two (Table 6) was not analyzed at 5 min so that theproduct could be purified by preparative HPLC. Nevertheless, the resultsdemonstrate that two PNA oligomer blocks, one with a N-terminal glycinecomprising a free N-terminal amine group and the other comprising aC-terminal glycine having a carboxylic acid functional group can beligated rapidly and efficiently with a mixture of HOAt and EDC tothereby produce an unmodified gly-gly dimer that links the two PNAoligomer blocks. The product of the ligation reaction wasAc-ACC-AG-gly-gly-TCC-AA-NH₂. This was confirmed by Maldi-TOF massspectrometry analysis of the final product.

Example 3 PNA-FISH

Introduction & Purpose

Prior experiments performed by Applicants have determined that certainprobes will not definitively distinguish single point mutations infunctional assays. For example, the PNA oligomer Flu-1 will notdefinitively distinguish between S. enterica (of which S. cholerasuis isa serovar) and S. bongori, despite the presence of a single pointmutation (single nucleotide polymorphism or SNP) that exists in the rRNAof these two organisms.

TABLE 7 Table Of Combination Oligomers & Probes Probe ID PNA ProbeSequence S/N Flu-1 F-OEE-ACC-TAC-GTG-TCA-GCG-EE-NH₂ 2.1 Flu-2F-OEE-TAC-GTG-TCA-GCG-TG-EE-NH₂ 5.3 Flu-3F-OEE-TAC-GTG-T-O-CAG-CGT-G-EE-NH₂ — Flu-4F-OEE-TAC-GTG-T-Gly-Gly-CAG-CGT-G-EE-NH₂ —

All abbreviations are as previously defined (See Table 2). The positionof the mismatch (single point mutation) in the rRNA or the Salmonellaspecies to be distinguished in this assay are in bold text. Signal tonoise ratios were obtained using an average based on single from 8bacteria.

PNA-FISH Procedure:

Individual 3 mL cultures of bacteria were grown overnight in Tryptic SoyBroth (TSB) at 30° C. The broth was then analyzed for absorbance at 600nm and then diluted into fresh TSB until the absorbance at 600 nm was0.5 OD/mL. These diluted culture stocks were then allowed to double 3-4times before harvesting. Cells from a 20 mL culture were pelleted bycentrifugation at 10,000 rpm for 5 minutes, resuspended in 20 mL PBS,pelleted again and resuspended in Fixation Buffer (4 % paraformaldehydein PBS (7 mM Na₂HPO₄; 3 mM NaH₂PO₄; 130 mM NaCl)). The bacteria wereincubated at room temperature for 30-60 minutes before they werepelleted again (centrifugation at 10,000 rpm for 5 minutes). Afterremoval of the fixation solution, the cells were resuspended in 20 mL of50 % aqueous ethanol. The fixed bacteria were then used after 30 minutesof incubation or optionally stored at −20° C. for up to several weeksbefore being used in an experiment.

For each sample prepared, 100 μL of fixed cells in 50% aqueous ethanolwas removed and centrifuged at 10,000 R.P.M. for 2 min. The ethanol wasthen remove from the sample and the pellet was resuspended in 100μL ofsterile PBS and pelleted again by centrifugation at 10,000 rpm for 2min.

The PBS was then removed from the pellet, and the cells were resuspendedin 100 μL of hybridization buffer (20 mM Tris-HCl, pH 9.0; 100 mM NaCl;0.5% SDS) that contained the appropriate probe (e.g. Flu-1 though Flu-4)at a concentration of 40 pmol/mL. The hybridization was performed at 55°C. for 30 minutes.

The sample was then centrifuged at 10,000 R.P.M. for 2 min. Thehybridization buffer was removed and the cells resuspended in 500 μLsterile TE-9.0 (10 mM Tris-HCl, pH 9.0; 1 mM EDTA). The solution wasallowed to stand at 55° C. for 5 minutes. The sample was thencentrifuged at 10,000 rpm for 5 minutes. The TE-9.0 was then removedfrom the pellet. The TE-9.0 wash was then repeated two more times.

After the final wash the cells were resuspended in 100 μL PBS. Analiquot of 2 μL of this suspension of cells was placed on a glass slide,spread and allowed to dry. Next, 1-2 μL of Vectashield (VectorLaboratories, P/N H-1000) and DAPI counterstain was deposited over thedried cells. A coverslip was added to the slide and its position fixedusing a couple of drops of nail polish. Each of the two differentbacterial strains, Salmonella cholerasuis and Salmonella bongori, werefixed and hybridized with each of probes Flu-1 through Flu-4 followingthe protocol described above. After hybridization and wash, each strainwas spotted and mounted on a microscope slide (see above) and examinedusing a Nikon fluorescent microscope equipped with a 60× immersion oilobjective, a 10× ocular (total enlargement is 600 fold), and an OmegaOptical XF22 filter.

Results

The panels in FIG. 2 are color images taken with a CCD camera equippedmicroscope using a two second exposure time. With reference to FIG. 2,panels A1-D1 are images taken of a PNA-FISH experiment using S.cholerasuis (a serovar of S. enterica) and the PNA probes or combinationoligomers Flu-1 to Flu-4, respectively. Similarly, panels A2-D2 areimages taken of a PNA-FISH experiment using S. bongori and the PNAprobes or combination oligomers Flu-1 to Flu-4, respectively.

With reference to FIG. 2, a comparison of panels A1 and A2 indicatesthat although the signal for S. cholerasuis is quite strong, the S.bongori is likewise detectable, albeit not as intensely positive.Therefore, this result indicates that the probe Flu-1 is not as highlydiscriminating as would be desirable despite there being a single pointmutation in the rRNA of the two Salmonella species. In addition to thedigital images, a signal to noise ratio was scored at 2.1 (Table 7).

With the probe Flu-2, the positioning of the mismatch was relocated tobe more terminally located as compared with being centrally located. Theprobe was also reduced in size from a 15-mer to a 14-mer (nucleobasesequence). With reference to Panels B1 and B2, it is evident that againthe signal for the S. cholerasuis is quite strong, while the signal forS. bongori is weaker, albeit still detectable. In addition to thedigital images, a signal to noise ratio was scored at 5.3 (Table 7).Taken together, the data indicates that moving the mismatch to a moreterminal position improves the discrimination of the assay.

The combination oligomers Flu-3 and Flu-4 utilize the same positioningof the mismatch as did Flu-2, and differ from each other only in thenature, but not the position, of the linker; the combination oligomerFlu-3 having a single O-linker bridge whereas the combination oligomerFlu-4 has the preferred gly-gly dimer bridge. With reference to panelsC1 and C2 in combination with D1 and D2, it is clear that although thesignal is weaker, under identical conditions, with the combinationoligomers (Flu-3 and Flu-4), the level of discrimination has improved ascompared with the native oligomer probe, Flu-2. Moreover, the signal tonoise level was so high, it could not be determined since there was novisible non-specific signal (noise) to count, and dividing by zero wouldbe meaningless.

Summary:

The discrimination between S. enterica serovar cholerasuis and S.bongori was greater with probes Flu-2, Flu-3 and Flu-4. Generally, theprobes Flu-2, Flu-3 and Flu-4 exhibited an excellent discriminationbetween the two species of Salmonella. However, by introducing the useof a combination oligomer (e.g. Flu-3 and Flu-4). even greater singlepoint mutation discrimination was possible as compared with nativeoligomers. Thus, the results demonstrate that the combination oligomersact in a more binary fashion (either hybridized or unhybridized) ascompared with native oligomers.

Example 4 Cleavage Assay

Introduction & Purpose:

This experiment was performed to determine whether or not a combinationoligomer comprising a cleavage site that was a substrate for an enzymewould be more or less apt to be cleaved by the operation of the enzymedepending on whether or not is was bound to a binding partner. Acombination oligomer labeled as a Linear Beacon was used since it wasenvisioned that both hybridization and cleavage data could bedetermined.

Materials:

The following PNA combination oligomers were synthesized usingcommercially available reagents and instrumentation (Applied Biosystems,Foster City, Calif.). These probes were designed to have lowfluorescence in the unhybridized state, medium fluorescence in thehybridized state and greatest fluorescence in the cleaved state.

PNA-Glu: F-O-ACCAG-Gly-Glu-TCCAA-K(D) PNA-Lys:F-O-ACCAG-Gly-Lys-TCCAA-K(D) (abbreviations have been previouslydefined; see Table 1)

The probes were purified by reversed-phase chromatography, and productfractions were identified by MALDI-TOF mass spectrometry, productfractions were combined and lyophilized. The dried material wasdissolved in 50% aqueous DMF at a concentration of ˜50 AU 260 units permL.

An oligonucleotide target, DNA Q of Example 1, was obtained from SigmaGenosys (Woodlands, Tex.) dissolved in water at a concentration of 365μM. Proteases were purchased from Roche Biochemicals, (Indianapolis,Ind.) and were formulated as follows:

-   Glu-C (Roche Cat. No. 1,420,399) was dissolved in Glu-C Buffer at a    concentration of 0.1 μg/μL.-   Glu-C Buffer was 25 mM ammonium carbonate, pH 7.8, containing 5% v/v    acetonitrile.-   Lys-C (Roche Cat. No. 1,420,429) was dissolved in Lys-C Buffer at a    concentration of 0.1 μg/μL.-   Lys-C Buffer was 25 mM Tris-HCl, 1 mM EDTA, pH 8.5, containing 5%    v/v acetonitrile.-   Trypsin (Roche Cat. No. 1,418,475) was dissolved in Trypsin Buffer    at a concentration of 0.1 μg/μL. Trypsin Buffer was 100 mM Tris-HCl,    pH 8.5, containing 5% v/v acetonitrile.    Methods:

Reactions were set up in a microtiter plate as follows:

PNA-Glu was diluted 1:2000 in Glu-C buffer. To 100 μL aliquots of thissolution in the wells of a microtiter plate was added:

-   -   PNA-Glu only    -   One μL of DNA Q    -   Two μL of Glu-C protease    -   One μl of DNA R, two μL of Glu-C protease

The timing and order of addition was firstly PNA-Glu in Glu-C Buffer(all wells A through D), followed by DNA Q (wells B and D), then a fiveminute wait, then Glu-C protease (wells C and D). After each additionreactions were mixed thoroughly. Reaction fluorescence was then measuredat 520 nm using a Wallac Victor plate reader (Wallac Oy, Turku Finland).Fluorescence was measured at time zero (after last addition) and twohours later.

Identical reactions were set up for PNA-Lys except that Lys-C Buffer andTrypsin Buffer was substituted for Glu-C Buffer, and Lys-C and Trypsinproteases were substituted for Glu-C protease, respectively.

Results:

The results are graphically illustrated in FIG. 3. In the absence of DNAtarget each protease was able to hydrolyze its respective PNA substrate(compare A and C in each set) whereas in the presence of target (DNA Q)the PNA was protected from digestion by the proteases (compare B and Din each set). Thus, the ability to discriminate between hybridized andunhybridized PNA combination oligomer with a protease was establishedsince the enzyme cleaved the cleavage site only if the combinationoligomer was not bound to the target sequence.

Example 5 SNP Scoring Using Independently Detectable Self-IndicatingCombination Oligomers and End-Point Analysis

Materials and Methods

SNP selection. Information regarding SNP targets, human genomic sequenceinformation, human genomic DNA samples, PCR primer designs, and SNP testresults obtained via the TaqMan Assay (Applied Biosystems, Foster City,Calif.) were provided by the Whitehead Institute (Cambridge, Mass.). Inview of the information provided, nine SNP targets, designated as 6874,6802, 6806, 6834, 6837, 6848, 6876, 6879, and 6885, were chosen forevaluation.

DNA samples. The CEPH/Utah Pedigrees 1331, 1333, and 1341 (hereinafter“Coriell Pedigree” or “Pedigree”), consisting of purified, human genomicDNA samples of related individuals, were purchased from CoriellInstitute for Medical Research (Camden, N.J.). Pedigree 1331 contained17 DNA samples designated as F01, M02, D03, S04, D05, D06, D07, D08,S09, S10, S11, FF12, FM13, MF14, MM15, D16, and S17. Pedigree 1333contained 15 DNA samples designated as F01, M02, S03, S04, S05, D06,S07, S08, S09, S10, FF11, FM12, MF13, MM14, and D15. Pedigree 1341contained 14 DNA samples designated as F01, M02, D03, D04, D05, D06,S07, D08, D09, S10, FF11, FM12, MF13, and MM14. The DNA samples, assupplied by the Coriell Institute, were diluted to 25 ng/μL in MilliporeMilli-Q water (Bedford, Mass.) and stored at 4° C.

PCR primers. The PCR primer designs offered by the Whitehead Institutewere all modified by sequence deletion, sequence addition, and/oraddition of guanine and/or cytosine bases at the 5′ end. Modification ofthe primers was performed so that unequal Tm values, of approximately66° C. and 80° C. for the two primers of the set as predicted at 200 nMconcentration, were obtained so that the PCR protocol produced bothdouble stranded and single stranded DNA amplicons with lower and higherannealing temperatures, respectively. The Tm predictions for primerdesign were performed using proprietary software based upon DNAnearest-neighbor parameters for predicting duplex stability as publishedby SantaLucia et al. Biochemistry 35: 3555-3562 (1996).

Once designed, all primers were purchased from Sigma Genosys (TheWoodlands, Tex.). The primers, as received from Sigma Genosys, werediluted to approximately 200 μM in 1× TE, pH 8.0(10 mM Tris, pH 8.0; 1mM EDTA) and stored at 4° C. One M Tris, pH 8.0 and EDTA disodium salt,dihydrate (used to make the storage buffers), were purchased from Sigma(St. Louis, Mo.) and EM Science (Gibbstown, N.J.), respectively.Properties of the nine primer sets are described in Table 8.

TABLE 8 Oligonucleotide PCR Primer Sets For SNP Analysis LengthPredicted Seq. SNP Name (bases) Tm (° C.) Sequence Id No. 6784 6784-5 2467.1 ACAAGTCTGGAGTGAGC 8 6784-3 33 81.1 GCGTGGCAGAGATCCCTGTTGC 9 68026802-5 22 81.0 GCCTCTGCAGGGTGCTGTCTTG 10 6802-3 19 67.3AAATGTTGGCTGCCAACTA 11 6806 6806-5 17 66.9 CTTGGAGCATCGAGACT 12 6806-322 80.1 GCGTGGCCTGTTTGGAGGTCAA 13 6834 6834-5 16 66.2 GAGGAGTGGTGCTGAT14 6834-3 25 80.3 GCGGAGGCCATAGCAGAAGAGAAGA 15 6837 6837-5 23 66.6CCAAGATCTCCAAGTAAAATAAC 16 6837-3 31 79.4CGCGCCAATAAATGTAAATGGCACAAATCCA 17 6848 6848-5 20 65.7CCTCATCCAGATAATGTTGT 18 6848-3 33 78.8 GCGCGCAAGAAAATGAATTTTGGCATAAAAA19 CT 6876 6876-5 16 66.4 TTCCTCAGCAACCCTG 20 6876-3 24 79.8GCATAGTGGACCCCAAGTCACCAT 21 6879 6879-5 24 65.8 GGAAATTGAATTTACCTTTTCATT22 6879-3 33 79.8 GGCGACATTCAAGTTGGAATAGTTCTGAGAG 23 TA 6885 6885-5 2479.7 GGCCAGGACCTGTTTGTGACATGA 24 6885-3 22 67.3 TTTGCTCAATGTGAAATGTTGT25

PNA probes. All PNA oligomers were synthesized using commerciallyavailable reagents and instrumentation except for labeling reagentsand/or linkers, which were either supplied by Applied Biosystems or elseprepared as described elsewhere (See: U.S. Pat. No. 6,355,421 based onapplication Ser. No. 09/179,289; herein incorporated by reference). ThePNA probes were of design “Dye-NNNNN-Gly-Gly-NNNNN-Lys(Dabcyl)” wherethe dye was either fluorescein (Flu), Tam (short for Tamra)), Dye1, orDye2 (the structure of Dye1 & Dye 2 are illustrated in FIG. 33; Dye 2 isalso described in U.S. Pat. No. 6,221,604, herein incorporated byreference). In this configuration, the set of PNA oligomers fordetermining a particular SNP were independently detectableself-indicating combination oligomers (See Table 9).

Flu- and Tam-labeled PNA oligomers were synthesized de novo, whereas theDye1- and Dye2-labeled PNA oligomer probes were each synthesized as two5mer oligomer blocks that were subsequently condensed/ligated to therebyproduce a 10mer PNA combination oligomer probe. For each set of probes,the nucleobase directed to the SNP target was located in the middle ofeither the N-terminal 5mer or the C-terminal 5mer. The Flu- andTam-labeled probes were diluted to approximately 400 μM in 50% aqueousDMF, whereas the Dye1- and Dye2-labeled probes were diluted to 50 μM in50% aqueous NMP. DMF and NMP were purchased from J T Baker (Philipsburg,N.J.) and Applied Biosystems, respectively. The ten PNA probe sets aredescribed in Table 9. In all cases, the probes of a set wereindependently detectable.

TABLE 9 Combination Oligomer Probe Sets For SNP Analysis. The probenucleobases directed to the SNP targets are in bold, underlined print.Com- SNP Name Sequence ments 6784 6784-1-A-Flu Flu-TG ACC-Gly-Gly-AGCAA- Lys(Dabcyl)-NH₂ 6784-1-C-Tam Tam-TG GCC-Gly-Gly-AGCAA- Lys(Dabcyl)-NH₂ 6802 6802-1-A-Flu Flu-GAGGT-Gly-Gly-CAT GG- Lys(Dabcyl)-NH₂ 6802-2-G-Tam Tam-GAGGT-Gly-Gly-CA C GG-Lys(Dabcyl)-NH₂ 6806 6806-1-A-Flu Flu-TGGTC-Gly-Gly-AA A GA-Lys(Dabcyl)-NH₂ 6806-1-G-Tam Tam-TGGTC-Gly-Gly-AA G GA- Lys(Dabcyl)-NH₂6834 6834-2-A-Flu Flu-AGGTA-Gly-Gly-AA A GA- Lys(Dabcyl)-NH₂6834-2-G-Tam Tam-AGGTA-Gly-Gly-AA G GA- Lys(Dabcyl)-NH₂ 68376837-2-C-Flu Flu-AG G AC-Gly-Gly-AGGGG- Lys(Dabcyl)-NH₂ 6837-2-T-TamTam-AG A AC-Gly-Gly-AGGGG- Lys(Dabcyl)-NH₂ 6848 6848-1-A-Flu Flu-AG AAT-Gly-Gly-GAGAC- Lys(Dabcyl)-NH₂ 6848-1-C-Tam Tam-AG CAT-Gly-Gly-GAGAC- Lys(Dabcyl)-NH₂ 6876 6876-1-A-Flu Flu-CTGGG-Gly-Gly-TTA TA- Lys(Dabcyl)-NH₂ 6876-1-G-Tam Tam-TT G TA-Gly-Gly-ACCAC-Lys(Dabcyl)-NH₂ 6879 6879-1-C-Flu Flu-GGATA-Gly-Gly-GT C GG-Lys(Dabcyl)-NH₂ 6879-1-T-Tam Tam-GT T GG-Gly-Gly-GTGAA- Lys(Dabcyl)-NH₂6885 6885-1-C-Flu F1u-GCAAG-Gly-Gly-AC G AG- Lys(Dabcyl)-NH₂6885-1-T-Tam Tam-GCAAG-Gly-Gly-AC A AG- Lys(Dabcyl)-NH₂ 6885-1-T-Dye1-GCAAG-Gly-Gly-AC A AG- Li- Dye1 Lys(Dabcyl)-NH₂ gated PNA probe6885-1-C- Dye2-GCAAG-Gly-Gly-AC G AG- Li- Dye2 Lys(Dabcyl)-NH₂ gated PNAprobe

PCR protocol. The PCR was performed in triplicate for each sample usingan ABI Prism 7700 Sequence Detector (Applied Biosystems). A No TargetControl (NTC) (absence of human genomic DNA target) was also performedfor each SNP tested. The PCR mixture contained 2 mM MgCl₂, 1× GoldBuffer, 0.25 mM dNTP with dTTP, 0.04 U/μL AmpliTaq Gold DNA Polymerase,200 nM of each primer, 200 nM of each PNA probe, and 0.5 ng/μL humangenomic DNA target. Millipore Milli-Q water was used as the PCR mixturediluent. One exception to this formulation included the PCR mixture forSNP 6834, which contained 2000 nM of the primer with the higher Tmvalue. This would permit the further generation of single stranded DNAamplicons by the PCR. Fifty μL of each sample were then loaded into aMicroAmp Optical 96-Well Reaction Plate and covered with an OpticalAdhesive Cover. The MgCl₂, Gold Buffer, dNTP with dTTP, AmpliTaq GoldDNA Polymerase, MicroAmp Optical 96-Well Reaction Plates, and OpticalAdhesive Covers were purchased from Applied Biosystems. Thethermocycling consisted of one round of enzyme activation (95° C., 10min); 30 rounds of denaturation (95° C., 15 s), annealing (60° C., 30s), and extension (75° C., 15 s); and 15 rounds of denaturation (95° C.,15 s), annealing (70° C., 30 s), and extension (75° C., 15 s). The lowerand higher annealing temperatures favored the generation of double andsingle stranded DNA amplicons, respectively, because of the unequalprimer Tm values. Following the PCR, the single stranded DNA ampliconsserved as the targets for the PNA probes.

Detection of fluorescence. Following the PCR, the 50 μL samples weretransferred to a 96 well, 250 μL, black polystyrene, V bottom Uniplatepurchased from Whatman Polyfiltronics (Clifton, N.J.). The fluorescenceof the dye-labeled PNA probes in each sample was then measured at roomtemperature in a Wallac Victor 1420 Multilabel Counter with Flu, Cy3,Dye1, and Dye2 filter sets (Wallac Oy, Turku, Finland). The Cy3 filterset was suitable for measuring Tam fluorescence. The Dye1 filter setconsisted of a 460-40 excitation filter and 510-10 emission filter,whereas the Dye2 filter set consisted of a 530-25 excitation filter and590-20 emission filter (Wallac Oy).

Data analysis. Bar graphs were generated by subtracting the fluorescentsignal of the NTC from the fluorescent signal of each sample; the NTCthen provided a signal of zero. Allele distribution plots (scatterplots) were generated by dividing the fluorescent signal of the NTC fromthe fluorescent signal of each sample; the NTC then provided a ratio ofone. This ratio was referred to as the signal/noise ratio.

Results

SNP analysis with de novo synthesized, Flu- and Tam-labeled PNA probes.The bar graphs and allele distribution plots for each SNP evaluated aredisplayed in FIGS. 4-25. A DNA sample that displayed high signal of onedye and negligible signal of the other dye was described as ahomozygote. The SNP variant of the homozygote was then determined by thedye that displayed the high signal. For example, the DNA sample M02 ofpedigree 1333 was deemed a homozygote for SNP 6802 as high Flu signaland negligible Tam signal were displayed (FIGS. 6 and 7). The SNP siteof that target was also deemed to contain the nucleobase adenine as theFlu-labeled PNA probe contained the nucleobase thymine at the SNP site(Table 9). In contrast, the other state of homozygosity was for thetarget containing the nucleobase guanine since the probe contained thenucleobase cytosine at the SNP site (See Table 9) and would have beendetermined if the Tamra-labeled probe (6802-2-G-Tam) produced thepredominate signal.

DNA sample S03 of pedigree 1333 provided an ambiguous result for SNP6806 (FIGS. 8 and 9). However, one replicate of the triplicates testedprovided low signal (data not shown). If this outlier were removed, theSNP result would have been deemed a homozygote as detected by theTam-labeled PNA probe and is described as such.

A DNA sample that displayed signals of both dyes of roughly equivalentintensity was described as a heterozygote. For example, the DNA sampleF01 of pedigree 1333 was deemed a heterozygote for SNP 6802 as Flu andTam signals were of roughly equivalent intensities (FIGS. 6 and 7). TheSNP sites of those targets were also deemed to contain the nucleobasesadenine and guanine as the Flu- and Tam-labeled PNA probes contained thenucleobases thymine and cytosine at the SNP sites, respectively (Table9).

The SNP results are summarized in Table 10. A homozygotic DNA sample wasillustrated by two adjacent plus signs under the same SNP targetnucleobase heading, whereas a heterozygotic DNA sample was illustratedby single plus signs under both SNP target nucleobase headings. Theresults obtained in this study are in full agreement with the dataprovided by the Whitehead Institute for samples analyzed with the TaqManAssay of Applied Biosystems, Foster City, Calif. (See: shaded rows ofTable 10).

TABLE 10 Chart of SNP results.

SNP analysis with ligated, Dye1- and Dye2-labeled PNA probes. Thesequences of Dye1- and Dye2-labeled PNA probes for SNP 6885 correspondedto their Tam- and Flu-labeled counterparts, respectively (Table 10). TheCoriell Pedigree 1341 chosen for evaluation displayed the anticipatedzygosities (FIGS. 26 and 27) and these results were in full agreementwith the results generated with the Flu- and Tam-labeled PNA probes(FIGS. 24, 25 and Table 10). Therefore, the fluorophores of Dye1 andDye2 provided compatibility with the assay and signal upon hybridizationto target. Furthermore, the results recorded demonstrate that thecondensation/ligation process imposed no adverse consequences upon thefunction of the probes.

Example 6 Ligation in the Absence of a Template Using Two LabeledOligomer Blocks

Introduction & Purpose

Example 2 it was demonstrated that ligation of unlabeled oligomer blockswas possible. This Example was intended to confirm whether or not it waspossible to ligate two labeled block oligomers and thereby produceindependently detectable, self-indicating combination oligomers.

Reagent Solutions:

-   -   0.75M 2-(N-Morpholino)ethane sulfonic acid (MES) Buffer; pH 6.0    -   35 mM HOBt Solution: Prepared by mixing 23.7 mg of        1-Hydroxybenzotriazole (HOBt) with 4.42 mL of 0.75M MES Buffer        pH 6.0.    -   EDC Solution was prepared by weighing out        1-Ethyl-3-(3-Dimethylamino-propyl)carbodiimide hydrochloride        (EDC) then adding a volume of the HOBt Solution prepared above        so that the final solution will deliver 0.8 mg EDC per 15 μL of        this solution. (EDC Solution is thus 0.278M)    -   Quenching Solution was prepared by adding 0.5 ml Trifluoroacetic        acid to 49.5 mL de-ionized water.    -   PNA Solution was prepared by dissolving PNA oligomer blocks in        50% Acetonitrile:50% 0.1% aqueous Trifluoroacetic acid to a        concentration of 2625 μM for the dye labeled block and 3000 μM        for the Dabcyl-labeled block.        General Reaction Conditions:

Final reaction conditions are generally 10 equivalents of HOBt and ˜160equivalents of EDC in a reaction mixture that was approximately 750 μMin each dye labeled oligomer block to be ligated. It is noted that inthese experiments, no template was used (i.e. non-template directedligation).

Experiment One:

About 5 μL of PNA Solution of each PNA oligomer block (the two oligomerblocks to be ligated) was added to a single 0.5 mL microcentrifuge tube.To the PNA oligomer mixture was added 7.5 μL of the EDC Solutionprepared as above. This solution was mixed by vortex and then placed ina heat block maintained at 80° C. After 60 min, 20 μL of the QuenchingSolution was added. The product was then analyzed by Maldi-TOF massspectrometry and HPLC.

Experiment Two:

About 5 μL of PNA Solution of each PNA oligomer block (the oligomerblocks to be ligated) was added to a single 0.5 mL microcentrifuge tube.To the PNA oligomer mixture was added 7.5 μL of the EDC Solutionprepared as above. This solution was mixed by vortex and then placed ina heat block maintained at 80° C. After 60 min, 20 μL of the QuenchingSolution was added. The product was then analyzed by Maldi-TOF massspectrometry and HPLC.

Results:

TABLE 11 Table Of Ligation Results Experiment # % Product (1 hour) 1 61*2 66* *Average of two ligations with different oligomer blocks

For both experiments the amine terminal block (condensing block) waslabeled with the dye (Dye1 or Dye2) and the carboxyl terminal block(terminal block) was labeled with dabcyl. Specifically, the condensingblock (PNA) was Dye1-TGG-TC-Gly-COOH and the terminal block wasH-Gly-AAA-GA-Lys(Dabcyl)-NH₂ for experiment one and Dye2-TGG-TC-Gly withterminal block Gly-AAG-GA-Lys(Dabcyl)-NH₂ in experiment two whereinLys(Dabcyl) represents a Dabcyl moiety attached to the γ amine of theamino acid Lysine, Gly represents that amino acid glycine and otherabbreviations are well known in the field of peptide chemistry.

With reference to Table 11, the percent completion of the ligationreaction was measured based on the integration of peak area (excludingthe peak representing the HOBt) of the HPLC analysis of the product. Theresults demonstrated that the two PNA oligomer blocks can, in theabsence of a template, be successfully ligated in greater than 50% yieldwithin 1 hour. The results demonstrate that two PNA oligomer blocks, onewith a N-terminal glycine comprising a free N-terminal amine group andthe other comprising a C-terminal glycine having a carboxylic acidfunctional group can be ligated rapidly and efficiently with a mixtureof HOBt and EDC to thereby produce an unmodified gly-gly dimer thatlinks the two PNA oligomer blocks. The products of the ligationreactions were Dye1-TGG-TC-gly-gly-AAA-GA-Lys(Dabcyl)-NH₂ andDye2-TGG-TC-gly-gly-AAG-GA-Lys(Dabcyl)-NH₂. This was confirmed byMaldi-TOF mass spectrometry analysis of the final product. Accordingly,the Example confirms that it is possible to ligate two labeled oligomerblocks to thereby produce independently detectable, self-indicatingcombination oligomers using a library approach.

Having described preferred embodiments of the invention, it will nowbecome apparent to one of skill in the art that other embodimentsincorporating the concepts may be used. It is felt, therefore, thatthese embodiments should not be limited to disclosed embodiments butrather should be limited only by the spirit and scope of the invention.

1. A method for forming a terminal oligomer block and a condensingoligomer block from a bifunctional single set library, said methodcomprising: (a) providing a bifunctional single set library comprisingat least two oligomer blocks; (b) treating an oligomer block of thebifunctional single set library to thereby remove one or more protectinggroups and produce a terminal oligomer block; and (c) treating anoligomer block of the bifunctional single set library to removedifferent one or more protecting groups as compared with step (b) andthereby produce a condensing oligomer block.
 2. The method of claim 1further comprising condensing the terminal oligomer block and thecondensing oligomer block.
 3. The method of claim 2, whereincondensation of the terminal oligomer block with the condensing oligomerblock produces a combination oligomer comprising a linker of at leastthree atoms in length.
 4. The method of claim 1, wherein the oligomerblocks of the bifunctional single set library are peptide nucleic acidoligomer, PNA chimera or PNA combination oligomer.